Methods for expanding sars-cov2-antigen-specific t cells, compositions and uses related thereto

ABSTRACT

Provided herein are methods for preparing and characterizing SARS-co2 antigen specific immune cell cultures and preparations and methods of using the same in adoptive immunotherapy for cancer, infections and immune disorders. Also, provided are compositions and methods for generating immune cells expressing synthetic antigen binding receptors targeting SARS-cov2 and methods of use of these cells for the treatment and prevention of COVID-19. Also provided are compositions and methods for determining immune response to SARs-cov2 in a subject, detecting SARS-cov2, measuring cytotoxicity induced by SARS-cov2, and detecting the expression and cytotoxicity of synthetic antigen binding receptors targeting SARS-cov2.

CROSS REFERENCE TO RELATED APPLICATIONS

The application claims priority under 35 U.S.C. § 119 to U.S.Provisional Application Ser. No. 63/030,592, filed May 27, 2020, thedisclosures of which are incorporated herein by reference.

TECHNICAL FIELD

The disclosure provides methods for preparing and characterizingSARS-co2 antigen specific immune cell cultures and preparations andmethods of using the same in adoptive immunotherapy for cancer,infections and immune disorders.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

Accompanying this filing is a Sequence Listing entitled“Sequence-Listing_ST25.txt”, created on May 27, 2021 and having3,708,371 bytes of data, machine formatted on IBM-PC, MS-Windowsoperating system. The sequence listing is hereby incorporated herein byreference in its entirety for all purposes.

BACKGROUND

Adoptive immunotherapy involves implanting or infusing disease-specificand/or engineered T cells, such as antigen-specific cytotoxic cells(CTLs) and synthetic antigen binding receptor (SABR)-expressing T cells,into individuals with the aim of recognizing, targeting, and destroyingdisease-associated cells.

CARs are synthetic immune-receptors, which can redirect T cells toselectively kill tumor cells. To overcome some of the design limitationof conventional 2nd generation CARs, several alternative designs,collectively termed next generation CARs, have been described, includingAb-TCR (WO 2017/070608 A1 incorporated herein by reference), TCRreceptor fusion proteins or TFP (WO 2016/187349 A1 incorporated hereinby reference), Synthetic Immune Receptors (SIRS) (see, WO 2018/102795A1, incorporated herein by reference), Tri-functional T cell antigencoupler (Tri-TAC) (see, WO 2015/117229 A1, incorporated herein byreference) and zSIR (see WO2019232503, incorporated herein byreference). The term SABR as described herein comprise single chainimmune receptors (e.g., 1^(st), 2^(nd) and 3^(rd) generation syntheticantigen binding receptors, TFPs, Tri-TAC and the like) and multiplechain immune receptors (e.g., SIR, zSIR, cTCR, ab-TCRs, αβTFP, γdTFP,recombinant TCRs, SAR etc.). SABR are capable of recognizing theirtarget antigen in MHC-dependent and MHC-independent manner. SABRs aresynthetic receptors typically consisting of a targeting/binding moietythat is associated with one or more signaling domains in one or morefusion molecules.

The proliferation and persistence of SABR-T cells in vivo is animportant factor in treatment efficacy. To this end, we herein describemethods for the generation and/or production of autologous andallogeneic antigen-specific T cells (e.g., T cells specificallysensitized to detect, for example, SARS-cov2-associated antigen(s)), andhave also been engineered to express at least one functional syntheticantigen binding receptor directed against a disease-associated antigenof choice.

SUMMARY OF THE INVENTION

Provided herein are methods of generating allogeneic or autologous Tcells that express a T cell receptor that specifically binds to anantigen (e.g., a virus antigen such as an SARS-cov2 peptide) presentedon a major histocompatibility complex (MHC) and a synthetic antigenbinding receptor (SABR) that specifically binds to a target-cell antigenor cell surface marker (e.g., a cancer cell-associated antigen such asCD 19). In some embodiments, the antigen-specific T cells are generatedby incubating a sample comprising T cells (responder cells, e.g., a PBMCsample or T cells isolated therefrom) with antigen presenting cells(APCs, i.e., stimulator cells) presenting an antigen (e.g., viralpeptide) on an MHC (e.g., a class I MHC encoded by an allele that ispresent in the subject), thereby inducing proliferation ofantigen-specific responder T cells. Preferably, the antigen-specificresponder T cells are transduced with a viral vector comprising anucleic acid sequence encoding a SABR. Also provided herein are methodsfor inducing ex vivo proliferation of a population of SABR-expressingantigen-specific T cells, comprising culturing a population of isolatedT cells with antigen-presenting stimulator cells and transducing theresultant antigen-specific T cells with a viral vector comprising anucleic acid sequence encoding a SABR. In some embodiments, thetransduced T cells are cultured with the antigen-presenting stimulatorcells to induce proliferation of antigen-specific SABR T cells. Incertain other embodiments, the isolated T cells are transduced with aSABR-encoding viral vector prior to culturing with APCs. In yet furtherembodiments, the isolated T cells are cultured with APCs prior to andfollowing transduction with a SABR-encoding viral vector.

In some aspects, provided herein are ex vivo methods for enrichingcentral memory T cells. In certain embodiments, such methods compriseobtaining a sample of cells from a subject comprising CD3⁺ T cells andcontacting said CD3⁺ T cells with antigen-presenting stimulator cells.In preferred embodiments, the CD3⁺ T cells are isolated from the sampleprior to contacting the antigen-presenting stimulator cells by methodsknown in the art (e.g., positive selection of CD3⁺ cells from the sampleand/or negative selection by depletion of undesired cells or componentsfrom the sample). For example, and without limitation, such methodsinclude selection with anti-CD3 beads (e.g., magnetic beads), plasticadherence, elutriation, depletion of NK cells (e.g., using anti-CD56beads), and/or combinations thereof. In some such embodiments, the CD3⁺T cells are transduced with a viral vector encoding a synthetic antigenbinding receptor (SABR) before and/or after contact with theantigen-presenting stimulator cells. In some such embodiments theSABR-expressing, CD3⁺, antigen-specific T cells are cultured withantigen-presenting stimulator cells.

In some embodiments, the stimulator cells are made to present at leastone virus peptide antigen by incubating the intended stimulator cellswith one or more of the virus peptides. In some embodiments, the virusis SARS-cov2.

The disclosure also provides isolated nucleic acids, polypeptides andvectors encoding SABR, wherein the antigen specific domain of the SABRtargets the spike glycoprotein of SARS-cov2. The disclosure alsoprovides cells and cell populations (such as T cells, NK cells, iPSC)comprising vectors encoding nucleic acids encoding SABR, wherein theantigen specific domain of the SABR targets spike glycoprotein ofSARS-cov2.

The disclosure also provides isolated nucleic acids, isolatedpolypeptides, vectors and kits for a fast, economical, sensitive andspecific assay for detection of antibodies, antibody fragments andnon-immunoglobulin antigen binding domains targeting the Spikeglycoprotein of SARS-cov2.

DETAILED DESCRIPTION Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The term “antigen” or “Ag” refers to a molecule that provokes an immuneresponse. This immune response may involve either antibody production,or the activation of specific immunologically-competent cells, or both.

The term “antigen presenting cell” or “APC” refers to an immune systemcell such as an accessory cell (e.g., a B-cell, a dendritic cell, andthe like) that displays a foreign antigen complexed with majorhistocompatibility complexes (WIC's) on its surface.

An “antigen binding domain” or “antigen binding module” or “antigenbinding segment” or “antigen specific domain” (ASD) refers to apolypeptide or peptide that due to its primary, secondary or tertiarysequence, post-translational modifications and/or charge binds to anantigen with a high degree of specificity.

The term “Ab-TCR” or “AbTCR” refers to a next generation CAR platform asdescribed in WO 2017/070608 A1 which is incorporated herein byreference.

The term “accessory module” refers to any molecule that is expressed inan immune cell (e.g., T cell, e.g., SABR-T cell, e.g., CAR-T cell orTCR-T cell) to decrease, regulate or modify the activity of the immunecell.

As used herein, the term “backbone” or “architecture” refers to theconfiguration of the different components (e.g., antigen bindingdomains, hinge domains, transmembrane domains, signaling domains) thatcomprise different SABR (e.g., CAR, SIR, cTCR, Ab-TCR, TFP etc.) and anyaccessory module which is generally optional.

Table A1: Exemplary SABR architectures. First generation conventionalCARs (Conventional CAR I) have an intracellular signaling (ISD) domain(e.g. CD3z) and no costimulatory domain. The TCR fusion proteins (TFP)are another example of conventional CAR 1. Second generationconventional CARs (Conventional CAR 2 or CAR II) have one costimulatorydomain (e.g. 41BB or CD28) and an intracellular signaling (ISD) domain(e.g. CD3z). Third generation conventional CARs (Conventional CAR 3 orCAR III) have two costimulatory domains (e.g. 41BB and CD28) and anintracellular signaling (ISD) domain (e.g. CD3z). Ab-TCRs are duel chainreceptors incorporating a vL-linker-TCR domain (TCRD and a vH-linker-TCRdomain (TCRD) and have been described in PCT/US2016/058305. cTCRs aresingle chain, one-and-half, or double chain receptors consisting ofantigen binding domain derived from a vL and vH fragment that are fusedto one or more TCR constant chain (TCR-C) and result in activation of Tcell signaling. Exemplary configurations of cTCR are described inPCT/US2017/064379 or WO 2018/102795 A1. Synthetic immune receptors arenext generation SABR and are described in PCT/US2017/064379 or WO2018/102795 A1. zSIRs are double chain receptors comprising two CD3zchains or fragments thereof with optional linkers and are described inPCT/US2019/035096. SABR may target a single antigen, two antigens ormultiple antigens. SABR may target the single epitope of a singleantigen or two or more epitopes of one or more antigens. The presentdisclosure covers unispecific, bispecific, multispecific, uni-paratopic,bi-paratopic and multi-paratopic SABRs.

TABLE A1 Exemplary SABR Architectures 1 CAR 1 or CAR I ASD HR TMD ISD(including TFP) 2 CAR 2 (CAR II) ASD HR TMD CSD ISD 3 CAR 3 (CAR III)ASD HR TMD CSD-I CSD-II ISD 4 Ab-TCR vL-cL TCRD(1) 2A vH-CH1 TCRD (II) 5Double Chain vL TCR-C(1) 2A vH TCR-C (II) cTCR/SIR-1 6 Double ChainvL-linker CD3z 2A vH-linker CD3z zSIR 6 One & Half Chain TCR-C(1) 2A ASDTCR-C (II) cTCR/SIR-3

TABLE 1 SEQ ID NO Name of vector or component SEQ ID NO 1 pLenti-EF1a 11pLENTI-NLuc-AcV5-Blasticidin- Pa08 2 pLenti-EF1a-DWPRE 12pLENTI-Gluc-Flag-blast-B07 3 MSCV-Bgl2-AvrII-Bam-EcoR1-Xho- 13 PolyABstB1-Mlu-Sal-ClaI.I03 4 pCCLc-MNDU3-WPRE 14 PolyA 5pCCLc-MNDU3-Eco-Nhe-Sal- 15 PolyA WPRE 6 pCCLc-MNDU3-delta-WPRE 16 polyA7 EF1alpha_(EF1a)_Promoter_Variant 17 pCDNA3 8 pSBbi-Pur 18 DNA barcode11 9 MSCVhygro-GLuc-HA-G02 19 DNA barcode 12 10 MSCVpac-GLUC-R03 20 DNAbarcode 13

TABLE 2 NAME SEQ ID NO (DNA) SEQ ID NO (PRT) LucPPe-146-1H2 21 491LucPPe-133-1B2 22 492 LucPPe-78-0B10 23 493 LucPPe49-7C6A 24 494LucPpL-81-6G1 25 495 GLuc 26 496 NLuc 27 497 TLuc 28 498 MLuc7- 29 499M43L/M110L LoLuc 30 500 HtLuc 31 501 PaLuc1 32 502 PaLuc2 33 503 MpLuc134 504 McLuc1 35 505 MaLuc1 36 506 MoLuc1 37 507 MoLuc2 38 508 MLuc39 39509 PsLuc1 40 510 LoLuc1-3 41 511 HtLuc2 42 512 Renilla-Luc 43 513

TABLE 3 SEQ ID SEQ ID NAME NO (DNA) NO (PRT) CD8_Signal_Peptide 45 515CD8-SIGNAL-PEPTIDE 46 516 IgH_Signal_Peptide 47 517 IgH_Signal_Peptide48 518 (GGGGS)x3-Linker 56 526 (GGSG)7_Linker 57 527 (GGSG)7_Linker_2 58528 DDAKK_linker 59 529

TABLE 4 Exemplary Tags and Linkers Name of SEQ ID SEQ ID fragment NO(DNA) NO (PRT) G4S 61 531 G3Sx2 62 532 G4Sx2 63 533 G4Sx3 64 534Myc-(P)-TAG 65 535 MYC2-TAG 66 536 MYC4-TAG 67 537 V5-TAG 68 538

TABLE 5 Exemplary SABR components Name of fragment SEQ ID NO (DNA) SEQID NO (PRT) hTCR-alpha- 78 548 constant_X02883.1 hTCRa-WT 79 549hTCRa-CSDVP 80 550 hTCRa-opt2 81 551 hTCRa-opt3 82 552 hTCRa-T48C-opt 83553 hTCRa-T48C-opt1 84 554 hTCRa-SDVP 85 555 hTCRa- S61R 86 556hTCRa-SDVPR 87 557 hTCRa-SD 88 558 hTCRaECD- 89 559 CD3zECDTMCP-opt2mTCRa-opt 90 560 cTCRa-opt 91 561 hTCR-b1-constant- 92 562region_X00437.1 hTCR-b2-constant- 93 563 region_L34740 hTCRb-WT 94 564hTCRb-S57C-opt1 95 565 hTCRb-KACIAH 96 566 41BB-CP-opt2 134 604CD3zECDTM-28z-opt 135 605 CD3zECDTM-BBz-opt 136 606 CD3zECDTM-28z-opt2137 607 CD3zECDTM-BBz-opt2 138 608 TCRa-wt2-opt-6MD 139 609TCRb-wt2-opt-6MD 140 610 TCRg-6MD 141 611 TCRd-6MD 142 612 IgCL 143 613IgG1-CH1 144 614 IgG1-CH1-DeltaC 145 615 IgG1-CH1-Hinge 146 616IgG1-CH1-v2 147 617 IgG1-CH1-DeltaC-v2 148 618 IgG1-CH1-Hinge-v2 149 619IgG2-0C-CH1 150 620 IgG2-IC-CHI1 151 621 IgG3-CHI1 152 622

TABLE 6 Therapeutic Controls SEQ ID NO SEQ ID NO Name of fragment (DNA)(PRT) PuroR_Variant-(PAC) 190 660 BlastR 191 661 CNB30 192 662GMCSF-SP-tEGFR 193 663 tEGFRviii 194 664 tCD19 195 665 tBCMA 196 666 K13197 667 MC159 198 668 K13-opt 199 669 icaspase-9 200 670

TABLE 7A SARS-cov2 vL, vH and ScFv Name of fragment SEQ ID NO (DNA) SEQID NO (PRT) CDR1 CDR2 CDR3 SARS-cov2-vL SARScov2-CR3022-vL 213 683 960961 962 SARScov2-S-RBD-B38-vL 214 684 963 964 965 SARS-cov2-S-RBD-H4-vL215 685 966 967 968 SARS-cov2-vH SARScov2-CR3022-vH 217 687 970 971 972SARScov2-S-RBD-B38-vH 218 688 973 974 975 SARS-cov2-S-RBD-H4-vH 219 689976 977 978 SARS-cov2-scFv SARScov2-CR3022-scFv 221 691SARScov2-S-RBD-B38- 222 692 scFv SARS-cov2-S-RBD-H4-scFv 223 693

TABLE 7B SARS-cov2 SEQ ID NO SEQ ID NO Name of fragment (DNA) (PRT)SARS-cov2-Poly-epitopes and Polypeptides SARS-cov2-Spike-Glycoprotein-S2224 694 SARScov2-HLA-A0201-Polypeptides 225 695Ub-G76A-SARScov2-HLA-A0201-Polyepitopes 226 696SARScov2-HLA-DRB10401-Polyepitopes 227 697Ub-G76A-SARScov2-HLA-DRB10401-Polyepitopes 228 698CD8SP-StreptagII-R1-S-Protein-RBD-Mlu-mCD8-hinge-TM 229 699 SARS-cov2and Relevant Cellular Proteins SARS-cov2-Spike-Glycoprotein-1-RBD(S1-RBD) 230 700 SARS-cov2-Spike-Glycoprotein 231 701SARS-cov2-nucleocapside-phosphoprotein 232 702SARS-cov2-membrane-glycoprotein 233 703 orf3a 234 704 ACE2 235 705TMPRSS2 236 706 SARS-cov2-Spike-Glycoprotein-S1-ND1 237 707SARS-cov2-Spike-Glycoprotein-S1-ND2 242 712SARS-cov2-Spike-Glycoprotein-S2 224 694 MALIBU-Glo ReagentsCD8SP-hu-mROO5-1-(vL-vH)-GGSG-NLuc-4xFLAG-x2STREP- 238 708 8xHis-T2A-PACCD8SP-SARScov2-CR3022-(vL-vH)-GGSG-NLuc-4xFLAG- 239 709x2STREP-8xHis-T2A-PAC CD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-GGSG-NLuc-4xFLAG-240 710 x2STREP-8xHis-T2A-PACCD8SP-SARScov2-S-RBD-B38-(vL-vH)-GGSG-NLuc-4xFLAG- 241 711x2STREP-8xHis-T2A-PAC Topanga ReagentsCD19-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis-T2A-Pac 243 713CD33-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis-T2A-Pac 244 714CD8SP-BCMA-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis-T2A- 245 715 PacCD8SP-StreptagII-R1-S1-Protein-RBD-ECD-GGSG-NLuc-4xFlag- 246 7162xStreptag-8xHis-T2A-PacCD8SP-R1-S1-Protein-RBD-ECD-GGSG-NLuc-4xFlag-2xStreptag- 247 7178xHis-T2A-Pac S-Protein-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis 248 718SARS-cov2-nucleocapsid-GGS-NLuc-4xFlag-2xStreptag-8xHis- 249 719 T2A-PACorf3a-GGSG-NLuc-4xFlag-2xStreptag-8xHis 250 720ACE2-ECD-GGS-NLuc-4xFlag-2xStreptag-8xHis 251 721

TABLE 8 Exemplary SABR Targeting CD19 SEQ SEQ ID ID NO NO Name offragment (DNA) (PRT)CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-Myc-CD8TM- 253 723BBz-T2A-PACCD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-Myc-28z-T2A- 254 724PAC CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-Myc-28z 255 725CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-Myc-CD8TM- 256 726 BBzCD8SP-hu-mROO5-1-vH-Gly-Ser-Linker-vL-Myc-CD8TM-BBz 257 727CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-Myc-CD8TM-z- 258 728P2A-K13-FLAG-T2A-PACCD8SP-hu-mROO5-1-vL-[hTCRa-CSDVP]-F-F2A-SP-hu-mROO5-1-vH- 259 729[hTCRb-KACIAH]-F-P2A-PACCD8SP-hu-mROO5-1-vL-[hTCRb-KACIAH]-F-P2A-SP-hu-mROO5-1-vH- 260 730[hTCRa-CSDVP]-F-F2A-PACCD8SP-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-SP-hu-mROO5-1-vH- 261 731[hTCRa-T48C] CD8SP-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-SP-hu-mROO5-1-vH-262 732 [hTCRa-T48C]-F-F2A-K13-optCD8SP-hu-mROO5-1-vL-[hTCRa-T48C]-F-P2A-SP-hu-mROO5-1-vH- 263 733[hTCRb-S57C] CD8SP-hu-mROO5-1-vL-[hTCRa-T48C]-F-P2A-SP-hu-mROO5-1-vH-264 734 [hTCRb-S57C]-F-P2A-K13-optCD8SP-V5-[hTCRb-KACIAH]-F-P2A-CD8SP-hu-mROO5-1-vL-Gly-Ser- 265 735Linker-hu-mROO5-1-vH-Myc-[hTCRa-CSDVP]-F-F2A-PACCD8SP-MYC-[hTCRa-T48C-opt1]-F-F2A-SP-hu-mROO5-1-vL-Gly-Ser- 266 736Linker-hu-mROO5-1-vH-V5-[hTCRb-S57C-opt1]-F-P2A-PACCD8SP-hu-mROO5-1-vL-[hTCRb-opt2]-F-P2A-SP-hu-mROO5-1-vH- 267 737[hTCRa-opt2]-F-F2A-PACCD8SP-hu-mROO5-1-vL-[hTCRb-opt2]-F-P2A-SP-hu-mROO5-1-vH-Myc- 268 738[preTCRa-Del48]-F-F2A-PACCD8SP-[hTCRb-opt2]-F-P2A-CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu- 269 739mROO5-1-vH-Myc4-[preTCRa-Del48]-F-F2A-PACCD8SP-hu-mROO5-1-vL-V5-[hTCRg1-opt]-F-P2A-SP-hu-mROO5-1-vH- 270 740Myc-[hTCRd-opt]-F-F2A-PACCD8SP-hu-mROO5-1-vL-[hTCRd-opt]-F-P2A-SP-hu-mROO5-1-vH- 271 741[hTCRg1-opt]CD8SP-V5-[hTCRg1-opt]-F-P2A-CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker- 272 742hu-mROO5-1-vH-Myc-[hTCRd-opt]-F-F2A-PACCD8SP-hu-mROO5-1-vL-IgCL-Bam-CD3zECDTMCP-opt-F-P2A-Spe-SP- 273 743Bst-hu-mROO5-1-vH-IgG1-CH1-KPN-CD3zECDTMCP-opt2-F-F2A-Xba- PACCD8SP-hu-mROO5-1-vL-[hTCRbECD-Bam-CD3zECDTMCP-opt]-F-P2A- 274 744SP-hu-mROO5-1-vH-[hTCRaECD-Kpn-CD3zECDTMCP-opt2]CD8SP-hu-mROO5-1-vL-[hTCRb-KAC-ECD-Bam-CD3zECDTMCP-opt]-F- 275 745P2A-SP-hu-mROO5-1-vH-[hTCRa-CSDVP-ECD-Kpn-CD3zECDTMCP- opt2]CD8SP-hu-mROO5-1-vL-V5-[hTCRbECD-Bam-CD3zECDTMCP-opt]-F- 276 746P2A-SP-hu-mROO5-1-vH-Myc-[hTCRaECD-Kpn-CD3zECDTM-28z-opt2]CD8SP-hu-mROO5-1-vL-V5-[hTCRbECD-Bam-CD3zECDTM-28z-opt]-F- 277 747P2A-SP-hu-mROO5-1-vH-Myc-[hTCRaECD-Kpn-CD3zECDTM-28z-opt2]CD8SP-hu-mROO5-1-vL-V5-[hTCRbECD-Bam-CD3zECDTMCP-opt]-F- 278 748P2A-SP-hu-mROO5-1-vH-Myc4-[hTCRaECD-Kpn-CD3zECDTM-BBz-opt2]CD8SP-hu-mROO5-1-vL-V5-[hTCRbECD-Bam-CD3zECDTM-BBz-opt]-F- 279 749P2A-SP-hu-mROO5-1-vH-Myc4-[hTCRaECD-Kpn-CD3zECDTM-BBz-opt2]CD8SP-hu-mROO5-1-vL-CD3zECDTMCP-opt-F-P2A-Spe-SP-Bst-hu- 280 750mROO5-1-vH-Mlu-CD3zECDTMCP-opt2-F-F2A-PACCD8SP-hu-mROO5-1-vL-[IgCL-TCRg-6MD]-F-P2A-SP-hu-mROO5-1-vH- 281 751[IgG1-CH1-TCRd-6MD]CD8SP-hu-mROO5-1-vL-[IgCL-TCRb-IAH-6MD]-F-P2A-SP-hu-mROO5-1- 282 752vH-[IgG1-CH1-TCRa-SDVP-6MD]CD8SP-hu-mROO5-1-vL-[IgCL-TCRb-wt-opt2-6MD]-F-P2A-SP-hu-mROO5- 283 7531-vH-[IgG1-CH1-TCRa-wt-op2-6MD]CD8SP-hu-mROO5-1-(vL-vH)-CD3e-ECDTMCP-opt2 284 754CD8SP-hu-mROO5-1-(vL-vH)-CD3d-ECDTMCP-opt2 285 755CD8SP-hu-mROO5-1-(vL-vH)-CD3g-ECDTMCP-opt2 286 756CD8SP-hu-mROO5-1-(vL-vH)-CD3z-ECDTMCP-opt2 287 757CD8SP-hu-mROO5-1-(vH-vL)-CD3e-ECDTMCP-opt2 288 758CD8SP-hu-mROO5-1-(vH-vL)-CD3d-ECDTMCP-opt2 289 759CD8SP-hu-mROO5-1-(vH-vL)-CD3g-ECDTMCP-opt2 290 760CD8SP-hu-mROO5-1-(vH-vL)-CD3z-ECDTMCP-opt2 291 761CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-[hTCRa-opt2]-F- 292 762F2A-PAC CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-[hTCRb-opt2]-F-293 763 F2A-PACCD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-Myc4-[preTCRa- 294 764Del48]-F-F2A-PAC CD8SP-hu-mROO5-1-vL-[hTCRb-opt2]-F-P2A-PAC 295 765CD8SP-hu-mROO5-1-vL-[hTCRb-opt2] 296 766IgHSP-hu-mROO5-1-vH-[hTCRa-opt2]-F-F2A-BlastR 297 767IgHSP-hu-mROO5-1-vH-[hTCRa-opt2] 298 768CD8SP-hu-mROO5-1-vL-V5-[hTCRb-S57C-opt]-F-P2A-PAC 299 769CD8SP-hu-mROO5-1-vL-V5-[hTCRb-S57C-opt] 300 770IgHSP-hu-mROO5-1-vH-Myc-[hTCRa-T48C-opt]-F-F2A-BlastR 301 771IgHSP-hu-mROO5-1-vH-Myc-[hTCRa-T48C-opt] 302 772CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-[hTCRa-SDVP]- 303 773F-F2A-PAC CD8SP-hu-mROO5-1-vL-Gly-Ser-Linker-hu-mROO5-1-vH-[hTCRb- 304774 KAIAH]-F-P2A-PACCD8SP-hu-mROO5-1-(vL-vH)-G4S-CD3e-ECDTMCP-opt2-F-F2A-PAC 305 775CD8SP-hu-mROO5-1-(vL-vH)-G4S-CD3d-ECDTMCP-opt2-F-F2A-PAC 306 776CD8SP-hu-mROO5-1-(vL-vH)-G4S-CD3g-ECDTMCP-opt2-F-F2A-PAC 307 777CD8SP-hu-mROO5-1-(vL-vH)-G4S-CD3z-ECDTMCP-opt2-F-F2A-PAC 308 778

TABLE 9 Exemplary SABR Targeting SARS-cov2 S-RBD SEQ SEQ ID ID NO NOName of fragment (DNA) (PRT)CD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-CD8TM- 309 779 BBzCD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-Myc- 310 780CD8TM-BBz-T2A-PACCD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-Myc-28z- 311 781T2A-PAC CD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-Myc-28z312 782 CD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-Myc- 313783 CD8TM-BBz CD8SP-SARScov2-CR3022-vH-GS-linker-vL-Myc-CD8TM-BBz 314784 CD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-Myc- 315 785CD8TM-z-P2A-K13-FLAG-T2A-PACCD8SP-SARScov2-CR3022-vL-[hTCRa-CSDVP]-F-F2A-SP-SARScov2- 316 786CR3022-vH-[hTCRb-KACIAH]-F-P2A-PACCD8SP-SARScov2-CR3022-vL-[hTCRb-KACIAH]-F-P2A-SP-SARScov2- 317 787CR3022-vH-[hTCRa-CSDVP]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-[hTCRb-S57C]-F-P2A-SP-SARScov2- 318 788CR3022-vH-[hTCRa-T48C]CD8SP-SARScov2-CR3022-vL-[hTCRb-S57C]-F-P2A-SP-SARScov2- 319 789CR3022-vH-[hTCRa-T48C]-F-F2A-K13-optCD8SP-SARScov2-CR3022-vL-[hTCRa-T48C]-F-P2A-SP-SARScov2- 320 790CR3022-vH-[hTCRb-S57C]CD8SP-SARScov2-CR3022-vL-[hTCRa-T48C]-F-P2A-SP-SARScov2- 321 791CR3022-vH-[hTCRb-S57C]-F-P2A-K13-optCD8SP-V5-[hTCRb-KACIAH]-F-P2A-CD8SP-SARScov2-CR3022-vL-GS- 322 792linker-SARScov2-CR3022-vH-Myc-[hTCRa-CSDVP]-F-F2A-PACCD8SP-MYC-[hTCRa-T48C-opt1]-F-F2A-SP-SARScov2-CR3022-vL-GS- 323 793linker-SARScov2-CR3022-vH-V5-[hTCRb-S57C-opt1]-F-P2A-PACCD8SP-SARScov2-CR3022-vL-[hTCRb-opt2]-F-P2A-SP-SARScov2- 324 794CR3022-vH-[hTCRa-opt2]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-[hTCRb-opt2]-F-P2A-SP-SARScov2- 325 795CR3022-vH-Myc-[preTCRa-Del48]-F-F2A-PACCD8SP-[hTCRb-opt2]-F-P2A-CD8SP-SARScov2-CR3022-vL-GS-linker- 326 796SARScov2-CR3022-vH-Myc4-[preTCRa-Del48]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-V5-[hTCRg1-opt]-F-P2A-SP-SARScov2- 327 797CR3022-vH-Myc-[hTCRd-opt]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-[hTCRd-opt]-F-P2A-SP-SARScov2- 328 798CR3022-vH-[hTCRg1-opt]CD8SP-V5-[hTCRg1-opt]-F-P2A-CD8SP-SARScov2-CR3022-vL-GS-linker- 329 799SARScov2-CR3022-vH-Myc-[hTCRd-opt]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-IgCL-Bam-CD3zECDTMCP-opt-F-P2A- 330 800Spe-SP-Bst-SARScov2-CR3022-vH-IgG1-CH1-KPN-CD3zECDTMCP-opt2-F-F2A-Xba-PAC CD8SP-SARScov2-CR3022-vL-[hTCRbECD-Bam-CD3zECDTMCP-opt]-F-331 801 P2A-SP-SARScov2-CR3022-vH-[hTCRaECD-Kpn-CD3zECDTMCP-opt2]CD8SP-SARScov2-CR3022-vL-[hTCRb-KAC-ECD-Bam-CD3zECDTMCP- 332 802opt]-F-P2A-SP-SARScov2-CR3022-vH-[hTCRa-CSDVP-ECD-Kpn- CD3zECDTMCP-opt2]CD8SP-SARScov2-CR3022-vL-V5-[hTCRbECD-Bam-CD3zECDTMCP- 333 803opt]-F-P2A-SP-SARScov2-CR3022-vH-Myc-[hTCRaECD-Kpn- CD3zECDTM-28z-opt2]CD8SP-SARScov2-CR3022-vL-V5-[hTCRbECD-Bam-CD3zECDTM-28z- 334 804opt]-F-P2A-SP-SARScov2-CR3022-vH-Myc-[hTCRaECD-Kpn- CD3zECDTM-28z-opt2]CD8SP-SARScov2-CR3022-vL-V5-[hTCRbECD-Bam-CD3zECDTMCP- 335 805opt]-F-P2A-SP-SARScov2-CR3022-vH-Myc4-[hTCRaECD-Kpn- CD3zECDTM-BBz-opt2]CD8SP-SARScov2-CR3022-vL-V5-[hTCRbECD-Bam-CD3zECDTM-BBz- 336 806opt]-F-P2A-SP-SARScov2-CR3022-vH-Myc4-[hTCRaECD-Kpn- CD3zECDTM-BBz-opt2]CD8SP-SARScov2-CR3022-vL-CD3zECDTMCP-opt-F-P2A-Spe-SP-Bst- 337 807SARScov2-CR3022-vH-Mlu-CD3zECDTMCP-opt2-F-F2A-PACCD8SP-SARScov2-CR3022-vL-[IgCL-TCRg-6MD]-F-P2A-SP-SARScov2- 338 808CR3022-vH-[IgG1-CH1-TCRd-6MD]CD8SP-SARScov2-CR3022-vL-[IgCL-TCRb-IAH-6MD]-F-P2A-SP- 339 809SARScov2-CR3022-vH-[IgG1-CH1-TCRa-SDVP-6MD]CD8SP-SARScov2-CR3022-vL-[IgCL-TCRb-wt-opt2-6MD]-F-P2A-SP- 340 810SARScov2-CR3022-vH-[IgG1-CH1-TCRa-wt-op2-6MD]CD8SP-SARScov2-CR3022-(vL-vH)-CD3e-ECDTMCP-opt2 341 811CD8SP-SARScov2-CR3022-(vL-vH)-CD3d-ECDTMCP-opt2 342 812CD8SP-SARScov2-CR3022-(vL-vH)-CD3g-ECDTMCP-opt2 343 813CD8SP-SARScov2-CR3022-(vL-vH)-CD3z-ECDTMCP-opt2 344 814CD8SP-SARScov2-CR3022-(vH-vL)-CD3e-ECDTMCP-opt2 345 815CD8SP-SARScov2-CR3022-(vH-vL)-CD3d-ECDTMCP-opt2 346 816CD8SP-SARScov2-CR3022-(vH-vL)-CD3g-ECDTMCP-opt2 347 817CD8SP-SARScov2-CR3022-(vH-vL)-CD3z-ECDTMCP-opt2 348 818CD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-[hTCRa- 349 819opt2]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-[hTCRP- 350 820opt2]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-Myc4- 351 821[preTCRa-Del48]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-[hTCRb-opt2]-F-P2A-PAC 352 822CD8SP-SARScov2-CR3022-vL-[hTCRb-opt2] 353 823IgHSP-SARScov2-CR3022-vH-[hTCRa-opt2]-F-F2A-BlastR 354 824IgHSP-SARScov2-CR3022-vH-[hTCRa-opt2] 355 825CD8SP-SARScov2-CR3022-vL-V5-[hTCRP-S57C-opt]-F-P2A-PAC 356 826CD8SP-SARScov2-CR3022-vL-V5-[hTCRP-S57C-opt] 357 827IgHSP-SARScov2-CR3022-vH-Myc-[hTCRa-T48C-opt]-F-F2A-BlastR 358 828IgHSP-SARScov2-CR3022-vH-Myc-[hTCRa-T48C-opt] 359 829CD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-[hTCRa- 360 830SDVP]-F-F2A-PACCD8SP-SARScov2-CR3022-vL-GS-linker-SARScov2-CR3022-vH-[hTCRP- 361 831KAIAH]-F-P2A-PACCD8SP-SARScov2-CR3022-(vL-vH)-G4S-CD3e-ECDTMCP-opt2-F-F2A- 362 832 PACCD8SP-SARScov2-CR3022-(vL-vH)-G4S-CD3d-ECDTMCP-opt2-F-F2A- 363 833 PACCD8SP-SARScov2-CR3022-(vL-vH)-G4S-CD3g-ECDTMCP-opt2-F-F2A- 364 834 PACCD8SP-SARScov2-CR3022-(vL-vH)-G4S-CD3z-ECDTMCP-opt2-F-F2A- 365 835 PAC

TABLE 10 Exemplary SABR Targeting SARS-cov2 S-RBD SEQ ID SEQ ID NO NOName of fragment (DNA) (PRT)CD8SP-SARScov2-S-RBD-H4-vL-GS-linker-SARScov2-S-RBD-H4-vH- 367 837Myc-CD8TM-BBz-T2A-PACCD8SP-SARScov2-S-RBD-H4-vL-GS-linker-SARScov2-S-RBD-H4-vH- 368 838Myc-28z-T2A-PACCD8SP-SARScov2-S-RBD-H4-vL-GS-linker-SARScov2-S-RBD-H4-vH- 369 839Myc-28z CD8SP-SARScov2-S-RBD-H4-vL-GS-linker-SARScov2-S-RBD-H4-vH- 370840 Myc-CD8TM-BBz CD8SP-SARScov2-S-RBD-H4-vH-GS-linker-vL-Myc-CD8TM-BBz371 841 CD8SP-SARScov2-S-RBD-H4-vL-GS-linker-SARScov2-S-RBD-H4-vH- 372842 Myc-CD8TM-z-P2A-K13-FLAG-T2A-PACCD8SP-SARScov2-S-RBD-H4-vL-[hTCRa-CSDVP]-F-F2A-SP- 373 843SARScov2-S-RBD-H4-vH-[hTCRb-KACIAH]-F-P2A-PACCD8SP-SARScov2-S-RBD-H4-vL-[hTCRb-KACIAH]-F-P2A-SP- 374 844SARScov2-S-RBD-H4-vH-[hTCRa-CSDVP]-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-[hTCRb-S57C]-F-P2A-SP-SARScov2- 375 845S-RBD-H4-vH-[hTCRa-T48C]CD8SP-SARScov2-S-RBD-H4-vL-[hTCRb-S57C]-F-P2A-SP-SARScov2- 376 846S-RBD-H4-vH-[hTCRa-T48C]-F-F2A-K13-optCD8SP-SARScov2-S-RBD-H4-vL-[hTCRa-T48C]-F-P2A-SP-SARScov2- 377 847S-RBD-H4-vH-[hTCRb-S57C]CD8SP-SARScov2-S-RBD-H4-vL-[hTCRa-T48C]-F-P2A-SP-SARScov2- 378 848S-RBD-H4-vH-[hTCRb-S57C]-F-P2A-K13-optCD8SP-V5-[hTCRb-KACIAH]-F-P2A-CD8SP-SARScov2-S-RBD-H4- 379 849vL-GS-linker-SARScov2-S-RBD-H4-vH-Myc-[hTCRa-CSDVP]-F-F2A- PACCD8SP-MYC-[hTCRa-T48C-opt1]-F-F2A-SP-SARScov2-S-RBD-H4-vL- 380 850GS-linker-SARScov2-S-RBD-H4-vH-V5-[hTCRb-S57C-opt1]-F-P2A- PACCD8SP-SARScov2-S-RBD-H4-vL-[hTCRb-opt2]-F-P2A-SP-SARScov2- 381 851S-RBD-H4-vH-[hTCRa-opt2]-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-[hTCRb-opt2]-F-P2A-SP-SARScov2- 382 852S-RBD-H4-vH-Myc-[preTCRa-Del48]-F-F2A-PACCD8SP-[hTCRb-opt2]-F-P2A-CD8SP-SARScov2-S-RBD-H4-vL-GS- 383 853linker-SARScov2-S-RBD-H4-vH-Myc4-[preTCRa-Del48]-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-V5-[hTCRg1-opt]-F-P2A-SP- 384 854SARScov2-S-RBD-H4-vH-Myc-[hTCRd-opt]-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-[hTCRd-opt]-F-P2A-SP-SARScov2-S- 385 855RBD-H4-vH-[hTCRg1-opt]CD8SP-V5-[hTCRg1-opt]-F-P2A-CD8SP-SARScov2-S-RBD-H4-vL-GS- 386 856linker-SARScov2-S-RBD-H4-vH-Myc-[hTCRd-opt]-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-IgCL-Bam-CD3zECDTMCP-opt-F- 387 857P2A-Spe-SP-Bst-SARScov2-S-RBD-H4-vH-IgG1-CH1-KPN-CD3zECDTMCP-opt2-F-F2A-Xba-PACCD8SP-SARScov2-S-RBD-H4-vL-[hTCRbECD-Bam-CD3zECDTMCP- 388 858opt]-F-P2A-SP-SARScov2-S-RBD-H4-vH-[hTCRaECD-Kpn- CD3zECDTMCP-opt2]CD8SP-SARScov2-S-RBD-H4-vL-[hTCRb-KAC-ECD-Bam- 389 859CD3zECDTMCP-opt]-F-P2A-SP-SARScov2-S-RBD-H4-vH-[hTCRa-CSDVP-ECD-Kpn-CD3zECDTMCP-opt2]CD8SP-SARScov2-S-RBD-H4-vL-V5-[hTCRbECD-Bam- 390 860CD3zECDTMCP-opt]-F-P2A-SP-SARScov2-S-RBD-H4-vH-Myc-[hTCRaECD-Kpn-CD3zECDTM-28z-opt2]CD8SP-SARScov2-S-RBD-H4-vL-V5-[hTCRbECD-Bam-CD3zECDTM- 391 86128z-opt]-F-P2A-SP-SARScov2-S-RBD-H4-vH-Myc-[hTCRaECD-Kpn-CD3zECDTM-28z-opt2] CD8SP-SARScov2-S-RBD-H4-vL-V5-[hTCRbECD-Bam- 392 862CD3zECDTMCP-opt]-F-P2A-SP-SARScov2-S-RBD-H4-vH-Myc4-[hTCRaECD-Kpn-CD3zECDTM-BBz-opt2]CD8SP-SARScov2-S-RBD-H4-vL-V5-[hTCRbECD-Bam-CD3zECDTM- 393 863BBz-opt]-F-P2A-SP-SARScov2-S-RBD-H4-vH-Myc4-[hTCRaECD-Kpn-CD3zECDTM-BBz-opt2]CD8SP-SARScov2-S-RBD-H4-vL-CD3zECDTMCP-opt-F-P2A-Spe-SP- 394 864Bst-SARScov2-S-RBD-H4-vH-Mlu-CD3zECDTMCP-opt2-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-[IgCL-TCRg-6MD]-F-P2A-SP- 395 865SARScov2-S-RBD-H4-vH-[IgG1-CH1-TCRd-6MD]CD8SP-SARScov2-S-RBD-H4-vL-[IgCL-TCRb-IAH-6MD]-F-P2A-SP- 396 866SARScov2-S-RBD-H4-vH-[IgG1-CH1-TCRa-SDVP-6MD]CD8SP-SARScov2-S-RBD-H4-vL-[IgCL-TCRb-wt-opt2-6MD]-F-P2A- 397 867SP-SARScov2-S-RBD-H4-vH-[IgG1-CH1-TCRa-wt-op2-6MD]CD8SP-SARScov2-S-RBD-H4-(vL-vH)-CD3e-ECDTMCP-opt2 398 868CD8SP-SARScov2-S-RBD-H4-(vL-vH)-CD3d-ECDTMCP-opt2 399 869CD8SP-SARScov2-S-RBD-H4-(vL-vH)-CD3g-ECDTMCP-opt2 400 870CD8SP-SARScov2-S-RBD-H4-(vL-vH)-CD3z-ECDTMCP-opt2 401 871CD8SP-SARScov2-S-RBD-H4-(vH-vL)-CD3e-ECDTMCP-opt2 402 872CD8SP-SARScov2-S-RBD-H4-(vH-vL)-CD3d-ECDTMCP-opt2 403 873CD8SP-SARScov2-S-RBD-H4-(vH-vL)-CD3g-ECDTMCP-opt2 404 874CD8SP-SARScov2-S-RBD-H4-(vH-vL)-CD3z-ECDTMCP-opt2 405 875CD8SP-SARScov2-S-RBD-H4-vL-GS-linker-SARScov2-S-RBD-H4-vH- 406 876[hTCRa-opt2]-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-GS-linker-SARScov2-S-RBD-H4-vH- 407 877[hTCRb-opt2]-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-GS-linker-SARScov2-S-RBD-H4-vH- 408 878Myc4-[preTCRa-Del48]-F-F2A-PACCD8SP-SARScov2-S-RBD-H4-vL-[hTCRb-opt2]-F-P2A-PAC 409 879CD8SP-SARScov2-S-RBD-H4-vL-[hTCRb-opt2] 410 880IgHSP-SARScov2-S-RBD-H4-vH-[hTCRa-opt2]-F-F2A-BlastR 411 881IgHSP-SARS-cov2-S-RBD-H4-vH-[hTCRa-opt2] 412 882CD8SP-SARS-cov2-S-RBD-H4-vL-V5-[hTCRb-S57C-opt]-F-P2A-PAC 413 883CD8SP-SARS-cov2-S-RBD-H4-vL-V5-[hTCRb-S57C-opt] 414 884IgHSP-SARS-cov2-S-RBD-H4-vH-Myc-[hTCRa-T48C-opt]-F-F2A- 415 885 BlastRIgHSP-SARS-cov2-S-RBD-H4-vH-Myc-[hTCRa-T48C-opt] 416 886CD8SP-SARS-cov2-S-RBD-H4-vL-Gly-Ser-Linker-SARS-cov2-S-RBD- 417 887H4-vH-[hTCRa-SDVP]-F-F2A-PACCD8SP-SARS-cov2-S-RBD-H4-vL-Gly-Ser-Linker-SARS-cov2-S-RBD- 418 888H4-vH-[hTCRb-KAIAH]-F-P2A-PACCD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-G4S-CD3e-ECDTMCP-opt2-F- 419 889F2A-PAC CD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-G4S-CD3d-ECDTMCP-opt2-F- 420890 F2A-PAC CD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-G4S-CD3g-ECDTMCP-opt2-F-421 891 F2A-PACCD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-G4S-CD3z-ECDTMCP-opt2-F- 422 892F2A-PAC

TABLE 11 Exemplary SABR Targeting SARS-cov2 S-RBD SEQ ID SEQ NO ID NOName of fragment (DNA) (PRT)CD8SP-SARScov2-S-RBD-B38-vL-GS-linker-SARScov2-S-RBD-B38-vH- 424 894Myc-CD8TM-BBz-T2A-PACCD8SP-SARScov2-S-RBD-B38-vL-GS-linker-SARScov2-S-RBD-B38-vH- 425 895Myc-28z-T2A-PACCD8SP-SARScov2-S-RBD-B38-vL-GS-linker-SARScov2-S-RBD-B38-vH- 426 896Myc-28z CD8SP-SARScov2-S-RBD-B38-vL-GS-linker-SARScov2-S-RBD-B38-vH- 427897 Myc-CD8TM-BBz CD8SP-SARScov2-S-RBD-B38-vH-GS-linker-vL-Myc-CD8TM-BBz428 898 CD8SP-SARScov2-S-RBD-B38-vL-GS-linker-SARScov2-S-RBD-B38-vH- 429899 Myc-CD8TM-z-P2A-K13-FLAG-T2A-PACCD8SP-SARScov2-S-RBD-B38-vL-[hTCRa-CSDVP]-F-F2A-SP- 430 900SARScov2-S-RBD-B38-vH-[hTCRb-KACIAH]-F-P2A-PACCD8SP-SARScov2-S-RBD-B38-vL-[hTCRb-KACIAH]-F-P2A-SP- 431 901SARScov2-S-RBD-B38-vH-[hTCRa-CSDVP]-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-[hTCRb-S57C]-F-P2A-SP-SARScov2- 432 902S-RBD-B38-vH-[hTCRa-T48C]CD8SP-SARScov2-S-RBD-B38-vL-[hTCRb-S57C]-F-P2A-SP-SARScov2- 433 903S-RBD-B38-vH-[hTCRa-T48C]-F-F2A-K13-optCD8SP-SARScov2-S-RBD-B38-vL-[hTCRa-T48C]-F-P2A-SP-SARScov2- 434 904S-RBD-B38-vH-[hTCRb-S57C]CD8SP-SARScov2-S-RBD-B38-vL-[hTCRa-T48C]-F-P2A-SP-SARScov2- 435 905S-RBD-B38-vH-[hTCRb-S57C]-F-P2A-K13-optCD8SP-V5-[hTCRb-KACIAH]-F-P2A-CD8SP-SARScov2-S-RBD-B38-vL- 436 906GS-linker-SARScov2-S-RBD-B38-vH-Myc-[hTCRa-CSDVP]-F-F2A-PACCD8SP-MYC-[hTCRa-T48C-opt1]-F-F2A-SP-SARScov2-S-RBD-B38-vL- 437 907GS-linker-SARScov2-S-RBD-B38-vH-V5-[hTCRb-S57C-opt1]-F-P2A-PACCD8SP-SARScov2-S-RBD-B38-vL-[hTCRb-opt2]-F-P2A-SP-SARScov2-S- 438 908RBD-B38-vH-[hTCRa-opt2]-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-[hTCRb-opt2]-F-P2A-SP-SARScov2-S- 439 909RBD-B38-vH-Myc-[preTCRa-Del48]-F-F2A-PACCD8SP-[hTCRb-opt2]-F-P2A-CD8SP-SARScov2-S-RBD-B38-vL-GS- 440 910linker-SARScov2-S-RBD-B38-vH-Myc4-[preTCRa-Del48]-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-V5-[hTCRg1-opt]-F-P2A-SP- 441 911SARScov2-S-RBD-B38-vH-Myc-[hTCRd-opt]-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-[hTCRd-opt]-F-P2A-SP-SARScov2-S- 442 912RBD-B38-vH-[hTCRg1-opt]CD8SP-V5-[hTCRg1-opt]-F-P2A-CD8SP-SARScov2-S-RBD-B38-vL-GS- 443 913linker-SARScov2-S-RBD-B38-vH-Myc-[hTCRd-opt]-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-IgCL-Bam-CD3zECDTMCP-opt-F- 444 914P2A-Spe-SP-Bst-SARScov2-S-RBD-B38-vH-IgG1-CH1-KPN-CD3zECDTMCP-opt2-F-F2A-Xba-PACCD8SP-SARScov2-S-RBD-B38-vL-[hTCRbECD-Bam-CD3zECDTMCP- 445 915opt]-F-P2A-SP-SARScov2-S-RBD-B38-vH-[hTCRaECD-Kpn- CD3zECDTMCP-opt2]CD8SP-SARScov2-S-RBD-B38-vL-[hTCRb-KAC-ECD-Bam- 446 916CD3zECDTMCP-opt]-F-P2A-SP-SARScov2-S-RBD-B38-vH-[hTCRa-CSDVP-ECD-Kpn-CD3zECDTMCP-opt2]CD8SP-SARScov2-S-RBD-B38-vL-V5-[hTCRbECD-Bam- 447 917CD3zECDTMCP-opt]-F-P2A-SP-SARScov2-S-RBD-B38-vH-Myc- zECDTM-28z-opt2]CD8SP-SARScov2-S-RBD-B38-vL-V5-[hTCRbECD-Bam-CD3zECDTM- 448 91828z-opt]-F-P2A-SP-SARScov2-S-RBD-B38-vH-Myc-[hTCRaECD-Kpn-CD3zECDTM-28z-opt2] CD8SP-SARScov2-S-RBD-B38-vL-V5-[hTCRbECD-Bam- 449919 CD3zECDTMCP-opt]-F-P2A-SP-SARScov2-S-RBD-B38-vH-Myc4-[hTCRaECD-Kpn-CD3zECDTM-BBz-opt2]CD8SP-SARScov2-S-RBD-B38-vL-V5-[hTCRbECD-Bam-CD3zECDTM- 450 920BBz-opt]-F-P2A-SP-SARScov2-S-RBD-B38-vH-Myc4-[hTCRaECD-Kpn-CD3zECDTM-BBz-opt2]CD8SP-SARScov2-S-RBD-B38-vL-CD3zECDTMCP-opt-F-P2A-Spe-SP- 451 921Bst-SARScov2-S-RBD-B38-vH-Mlu-CD3zECDTMCP-opt2-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-[IgCL-TCRg-6MD]-F-P2A-SP- 452 922SARScov2-S-RBD-B38-vH-[IgG1-CH1-TCRd-6MD]CD8SP-SARScov2-S-RBD-B38-vL-[IgCL-TCRb-IAH-6MD]-F-P2A-SP- 453 923SARScov2-S-RBD-B38-vH-[IgG1-CH1-TCRa-SDVP-6MD]CD8SP-SARScov2-S-RBD-B38-vL-[IgCL-TCRb-wt-opt2-6MD]-F-P2A-SP- 454 924SARScov2-S-RBD-B38-vH-[IgG1-CH1-TCRa-wt-op2-6MD]CD8SP-SARScov2-S-RBD-B38-(vL-vH)-CD3e-ECDTMCP-opt2 455 925CD8SP-SARScov2-S-RBD-B38-(vL-vH)-CD3d-ECDTMCP-opt2 456 926CD8SP-SARScov2-S-RBD-B38-(vL-vH)-CD3g-ECDTMCP-opt2 457 927CD8SP-SARScov2-S-RBD-B38-(vL-vH)-CD3z-ECDTMCP-opt2 458 928CD8SP-SARScov2-S-RBD-B38-(vH-vL)-CD3e-ECDTMCP-opt2 459 929CD8SP-SARScov2-S-RBD-B38-(vH-vL)-CD3d-ECDTMCP-opt2 460 930CD8SP-SARScov2-S-RBD-B38-(vH-vL)-CD3g-ECDTMCP-opt2 461 931CD8SP-SARScov2-S-RBD-B38-(vH-vL)-CD3z-ECDTMCP-opt2 462 932CD8SP-SARScov2-S-RBD-B38-vL-GS-linker-SARScov2-S-RBD-B38-vH- 463 933[hTCRa-opt2]-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-GS-linker-SARScov2-S-RBD-B38-vH- 464 934[hTCRb-opt2]-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-GS-linker-SARScov2-S-RBD-B38-vH- 465 935Myc4-[preTCRa-Del48]-F-F2A-PACCD8SP-SARScov2-S-RBD-B38-vL-[hTCRb-opt2]-F-P2A-PAC 466 936CD8SP-SARScov2-S-RBD-B38-vL-[hTCRb-opt2] 467 937IgHSP-SARScov2-S-RBD-B38-vH-[hTCRa-opt2]-F-F2A-BlastR 468 938IgHSP-SARS-cov2-S-RBD-B38-vH-[hTCRa-opt2] 469 939CD8SP-SARS-cov2-S-RBD-B38-vL-V5-[hTCRb-S57C-opt]-F-P2A-PAC 470 940CD8SP-SARS-cov2-S-RBD-B38-vL-V5-[hTCRb-S57C-opt] 471 941IgHSP-SARS-cov2-S-RBD-B38-vH-Myc-[hTCRa-T48C-opt]-F-F2A-BlastR 472 942IgHSP-SARS-cov2-S-RBD-B38-vH-Myc-[hTCRa-T48C-opt] 473 943CD8SP-SARS-cov2-S-RBD-B38-vL-Gly-Ser-Linker-SARS-cov2-S-RBD- 474 944B38-vH-[hTCRa-SDVP]-F-F2A-PACCD8SP-SARS-cov2-S-RBD-B38-vL-Gly-Ser-Linker-SARS-cov2-S-RBD- 475 945B38-vH-[hTCRb-KAIAH]-F-P2A-PACCD8SP-SARS-cov2-S-RBD-B38-(vL-vH)-G4S-CD3e-ECDTMCP-opt2-F- 476 946F2A-PAC CD8SP-SARS-cov2-S-RBD-B38-(vL-vH)-G4S-CD3d-ECDTMCP-opt2-F- 477947 F2A-PAC CD8SP-SARS-cov2-S-RBD-B38-(vL-vH)-G4S-CD3g-ECDTMCP-opt2-F-478 948 F2A-PACCD8SP-SARS-cov2-S-RBD-B38-(vL-vH)-G4S-CD3z-ECDTMCP-opt2-F- 479 949F2A-PAC

TABLE 12 SEQ SEQ ID NO ID NO Name of fragment (DNA) (PRT) TherapeuticControls F2A 481 951 T2A 482 952 P2A 483 953 E2A 484 954 SGSG 485 955FURINE-CLEAVAGE-SITE 486 956 FURINE-CLEAVAGE-SITE 487 957FURINE-CLEAVAGE-SITE 488 958 Miscellaneous FKBP12-F36V 1041 1161IKZF1-ZF2-145-167 1042 1162 IKZF1-ZF2-ZF3-145-197 1043 1163IKZF1-ZF2-ZF3-145-243 1044 1164 IKZFl-ZF2H1ZF2H2ZF2H3-ZF2H2x3 1045 1165SARS-cov2-S-Prt-F-P2A-Membrane-gp-F-F2A-Nucleocapsid-F-E2A-orf3 10471167 CD8SP-StreptagII-R1-S-Protein-RBD-Mlu-mCD8-hinge-TM-F-P2A- 10481168 Membrane-gp-F-F2A-NucleocapsidSARS-cov2-S-Prt-F-P2A-Membrane-gp-F-F2A-Nucleocapsid 1049 1169SARScov2-HLA-A0201-PEPTIDES-F-P2A-Membrane-gp-F-F2A- 1050 1170NucleocapsidUb-G76A-SARScov2-HLA-A0201-Polyepitopes-F-P2A-Membrane-gp-F- 1051 1171F2A-NucleocapsidSARScov2-HLA-DRB10401-Polyepitopes-F-P2A-Membrane-gp-F-F2A- 1052 1172NucleocapsidUb-G76A-SARScov2-HLA-DRB10401-Polyepitopes-F-P2A-Membrane-gp- 1053 1173F-F2A-NucleocapsidSARS-cov2-Spike-Glycoprotein-1-RBD-(S1-RBD)-FKBP12-F36V 1054 1174SARS-cov2-Spike-Glycoprotein-1-RBD-(S1-RBD)-IKZF1-ZF2-ZF3-145- 1055 1175243 SARS-cov2-Spike-Glycoprotein-FKBP12-F36V 1056 1176SARS-cov2-Spike-Glycoprotein-IKZF1-ZF2-ZF3-145-243 1057 1177SARS-cov2-nucleocapsid-FKBP12-F36V 1058 1178SARS-cov2-nucleocapsid-IKZF1-ZF2-ZF3-145-243 1059 1179SARS-cov2-membrane-Glycoprotein-FKBP12-F36V 1060 1180SARS-cov2-membrane-Glycoprotein-IKZF1-ZF2-ZF3-145-243 1061 1181CD8SP-StreptagII-R1-S-Protein-RBD-Mlu-mCD8-hinge-TM-F-P2A- 1062 1182Membrane-gp-F-F2A-Nucleocapsid-FKBP12-F36VCD8SP-StreptagII-R1-S-Protein-RBD-Mlu-mCD8-hinge-TM-F-P2A- 1063 1183Membrane-gp-F-F2A-Nucleocapsid-IKZF1-ZF2-145-167CD8SP-StreptagII-R1-S-Protein-RBD-Mlu-mCD8-hinge-TM-F-P2A- 1064 1184Membrane-gp-F-F2A-Nucleocapsid-IKZF1-ZF2-ZF3-145-197CD8SP-StreptagII-R1-S-Protein-RBD-Mlu-mCD8-hinge-TM-F-P2A- 1065 1185Membrane-gp-F-F2A-Nucleocapsid-IKZF1-ZF2-ZF3-145-243SARS-cov2-S-Prt-F-P2A-Membrane-gp-F-F2A-Nucleocapsid-FKBP12- 1066 1186F36V SARScov2-HLA-A0201-PEPTIDES-F-P2A-Membrane-gp-F-F2A- 1067 1187Nucleocapsid-FKBP12-F36VUb-G76A-SARScov2-HLA-A0201-Polyepitopes-F-P2A-Membrane-gp-F- 1068 1188F2A-Nucleocapsid-FKBP12-F36VSARScov2-HLA-DRB10401-Polyepitopes-F-P2A-Membrane-gp-F-F2A- 1069 1189Nucleocapsid-FKBP12-F36VUb-G76A-SARScov2-HLA-DRB10401-Polyepitopes-F-P2A-Membrane-gp- 1070 1190F-F2A-Nucleocapsid-FKBP12-F36VSARS-cov2-S-Prt-F-P2A-Membrane-gp-F-F2A-Nucleocapsid-IKZF1-ZF2- 10711191 ZF3-145-243 SARScov2-HLA-A0201-PEPTIDES-F-P2A-Membrane-gp-F-F2A-1072 1192 Nucleocapsid-IKZF1-ZF2-ZF3-145-243Ub-G76A-SARScov2-HLA-A0201-Polyepitopes-F-P2A-Membrane-gp-F- 1073 1193F2A-Nucleocapsid-IKZF1-ZF2-ZF3-145-243SARScov2-HLA-DRB10401-Polyepitopes-F-P2A-Membrane-gp-F-F2A- 1074 1194Nucleocapsid-IKZF1-ZF2-ZF3-145-243Ub-G76A-SARScov2-HLA-DRB10401-Polyepitopes-F-P2A-Membrane-gp- 1075 1195F-F2A-Nucleocapsid-IKZF1-ZF2-ZF3-145-243 1076 1196SARS-cov2-Spike-Glycoprotein-S2-GGSG-NLuc-4xFLAG-x2STREP- 1077 11978xHis-T2A-PACSARS-cov2-Spike-Glycoprotein-S1-ND1-GGSG-NLuc-4xFLAG-x2STREP- 1078 11988xHis-T2A-PACSARS-cov2-Spike-Glycoprotein-S1-ND2-GGSG-NLuc-4xFLAG-x2STREP- 1079 11998xHis-T2A-PAC CD8SP-StreptagII-R1-S-Protein-RBD-Mlu-mCD8-hinge-TM 10801200 CD8SP-hu-mROO5-1-(vL-vH)-GGSG-NLuc-4xFLAG-x2STREP-8xHis- 1081 1201T2A-PAC CD8SP-SARScov2-CR3022-(vL-vH)-GGSG-NLuc-4xFLAG-x2STREP- 10821202 8xHis-T2A-PACCD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-GGSG-NLuc-4xFLAG-x2STREP- 1083 12038xHis-T2A-PAC CD8SP-SARScov2-S-RBD-B38-(vL-vH)-GGSG-NLuc-4xFLAG-x2STREP-1084 1204 8xHis-T2A-PACCD19-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis-T2A-Pac 1085 1205CD33-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis-T2A-Pac 1086 1206CD8SP-BCMA-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis-T2A-Pac 1087 1207CD8SP-StreptagII-R1-S1-Protein-RBD-ECD-GGSG-NLuc-4xFlag- 1088 12082xStreptag-8xHis-T2A-PacCD8SP-R1-S1-Protein-RBD-ECD-GGSG-NLuc-4xFlag-2xStreptag- 1089 12098xHis-T2A-Pac S1-Protein-ECD-GGSG-NLuc-4xFlag-2xStreptag-8xHis 1090 1210SARS-cov2-nucleocapsid-GGS-NLuc-4xFlag-2xStreptag-8xHis-T2A-PAC 10911211 orf3a-GGSG-NLuc-4xFlag-2xStreptag-8xHis 1092 1212ACE2-ECD-GGS-NLuc-4xFlag-2xStreptag-8xHis 1093 1213SARS-cov2-Spike-Glycoprotein-S2-GGSG-Gluc-4xFLAG-x2STREP- 1095 12158xHis-T2A-PACSARS-cov2-Spike-Glycoprotein-S1-ND1-GGSG-Gluc-4xFLAG-x2STREP- 1096 12168xHis-T2A-PACSARS-cov2-Spike-Glycoprotein-S1-ND2-GGSG-Gluc-4xFLAG-x2STREP- 1097 12178xHis-T2A-PAC CD8SP-hu-mROO5-1-(vL-vH)-GGSG-Gluc-4xFLAG-x2STREP-8xHis-1098 1218 T2A-PACCD8SP-SARScov2-CR3022-(vL-vH)-GGSG-Gluc-4xFLAG-x2STREP- 1099 12198xHis-T2A-PAC CD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-GGSG-Gluc-4xFLAG-x2STREP-1100 1220 8xHis-T2A-PACCD8SP-SARScov2-S-RBD-B38-(vL-vH)-GGSG-Gluc-4xFLAG-x2STREP- 1101 12218xHis-T2A-PAC CD19-ECD-GGSG-Gluc-4xFlag-2xStreptag-8xHis-T2A-Pac 11021222 CD33-ECD-GGSG-Gluc-4xFlag-2xStreptag-8xHis-T2A-Pac 1103 1223CD8SP-BCMA-ECD-GGSG-Gluc-4xFlag-2xStreptag-8xHis-T2A-Pac 1104 1224CD8SP-StreptagII-R1-S1-Protein-RBD-ECD-GGSG-Gluc-4xFlag- 1105 12252xStreptag-8xHis-T2A-PacCD8SP-R1-S1-Protein-RBD-ECD-GGSG-Gluc-4xFlag-2xStreptag-8xHis- 1106 1226T2A-Pac S1-Protein-ECD-GGSG-Gluc-4xFlag-2xStreptag-8xHis 1107 1227SARS-cov2-nucleocapsid-GGS-Gluc-4xFlag-2xStreptag-8xHis-T2A-PAC 11081228 orf3a-GGSG-Gluc-4xFlag-2xStreptag-8xHis 1109 1229ACE2-ECD-GGS-Gluc-4xFlag-2xStreptag-8xHis 1110 1230SARS-cov2-Spike-Glycoprotein-S2-GGSG-TLuc-4xFLAG-x2STREP-8xHis- 11121232 T2A-PACSARS-cov2-Spike-Glycoprotein-S1-ND1-GGSG-TLuc-4xFLAG-x2STREP- 1113 12338xHis-T2A-PACSARS-cov2-Spike-Glycoprotein-S1-ND2-GGSG-TLuc-4xFLAG-x2STREP- 1114 12348xHis-T2A-PAC CD8SP-hu-mROO5-1-(vL-vH)-GGSG-TLuc-4xFLAG-x2STREP-8xHis-1115 1235 T2A-PACCD8SP-SARScov2-CR3022-(vL-vH)-GGSG-TLuc-4xFLAG-x2STREP- 1116 12368xHis-T2A-PAC CD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-GGSG-TLuc-4xFLAG-x2STREP-1117 1237 8xHis-T2A-PACCD8SP-SARScov2-S-RBD-B38-(vL-vH)-GGSG-TLuc-4xFLAG-x2STREP- 1118 12388xHis-T2A-PAC CD19-ECD-GGSG-TLuc-4xFlag-2xStreptag-8xHis-T2A-Pac 11191239 CD33-ECD-GGSG-TLuc-4xFlag-2xStreptag-8xHis-T2A-Pac 1120 1240CD8SP-BCMA-ECD-GGSG-TLuc-4xFlag-2xStreptag-8xHis-T2A-Pac 1121 1241CD8SP-StreptagII-R1-S1-Protein-RBD-ECD-GGSG-TLuc-4xFlag- 1122 12422xStreptag-8xHis-T2A-PacCD8SP-R1-S1-Protein-RBD-ECD-GGSG-TLuc-4xFlag-2xStreptag-8xHis- 1123 1243T2A-Pac S1-Protein-ECD-GGSG-TLuc-4xFlag-2xStreptag-8xHis 1124 1244SARS-cov2-nucleocapsid-GGS-TLuc-4xFlag-2xStreptag-8xHis-T2A-PAC 11251245 orf3a-GGSG-TLuc-4xFlag-2xStreptag-8xHis 1126 1246ACE2-ECD-GGS-TLuc-4xFlag-2xStreptag-8xHis 1127 1247SARS-cov2-Spike-Glycoprotein-S2-GGSG-MLuc7-M43L-M110L-4xFLAG- 1129 1249x2STREP-8xHis-T2A-PACSARS-cov2-Spike-Glycoprotein-S1-ND1-GGSG-MLuc7-M43L-M110L- 1130 12504xFLAG-x2STREP-8xHis-T2A-PACSARS-cov2-Spike-Glycoprotein-S1-ND2-GGSG-MLuc7-M43L-M110L- 1131 12514xFLAG-x2STREP-8xHis-T2A-PACCD8SP-hu-mROO5-1-(vL-vH)-GGSG-MLuc7-M43L-M110L-4xFLAG- 1132 1252x2STREP-8xHis-T2A-PACCD8SP-SARScov2-CR3022-(vL-vH)-GGSG-MLuc7-M43L-M110L- 1133 12534xFLAG-x2STREP-8xHis-T2A-PACCD8SP-SARS-cov2-S-RBD-H4-(vL-vH)-GGSG-MLuc7-M43L-M110L- 1134 12544xFLAG-x2STREP-8xHis-T2A-PACCD8SP-SARScov2-S-RBD-B38-(vL-vH)-GGSG-MLuc7-M43L-M110L- 1135 12554xFLAG-x2STREP-8xHis-T2A-PACCD19-ECD-GGSG-MLuc7-M43L-M110L-4xFlag-2xStreptag-8xHis-T2A- 1136 1256Pac CD33-ECD-GGSG-MLuc7-M43L-M110L-4xFlag-2xStreptag-8xHis-T2A- 11371257 Pac CD8SP-BCMA-ECD-GGSG-MLuc7-M43L-M110L-4xFlag-2xStreptag- 11381258 8xHis-T2A-PacCD8SP-StreptagII-R1-S1-Protein-RBD-ECD-GGSG-MLuc7-M43L-M110L- 1139 12594xFlag-2xStreptag-8xHis-T2A-PacCD8SP-R1-S1-Protein-RBD-ECD-GGSG-MLuc7-M43L-M110L-4xFlag- 1140 12602xStreptag-8xHis-T2A-PacS1-Protein-ECD-GGSG-MLuc7-M43L-M110L-4xFlag-2xStreptag-8xHis 1141 1261SARS-cov2-nucleocapsid-GGS-MLuc7-M43L-M110L-4xFlag-2xStreptag- 1142 12628xHis-T2A-PAC orf3a-GGSG-MLuc7-M43L-M110L-4xFlag-2xStreptag-8xHis 11431263 ACE2-ECD-GGS-MLuc7-M43L-M110L-4xFlag-2xStreptag-8xHis 1144 1264

As used herein, the term “biological equivalent thereof” or “variant” or“functional variant” is intended to be synonymous with “equivalentthereof” when referring to a reference protein, antibody or fragmentthereof, polypeptide or nucleic acid, intends those having minimalhomology while still maintaining desired structure or functionality.

As used herein, the term “CDR” or “complementarity determining region”is intended to mean the non-contiguous antigen combining sites foundwithin the variable region of both heavy and light chain polypeptides.The SEQ IDs of the CDRs of the different vL and vH segments that can beused in the construction of antigen binding domains of SABR targetingthe receptor binding domain of the spike glycoprotein of SARS-cov2 ofthe current disclosure are provided in Table 7A.

The SEQ IDs of the CDRs of the different vL and vH segments that can beused in the construction of antigen binding domains of SABR targetingdifferent antigens (e.g., CD19, CD20, CD22, Mesothelin etc.) of thecurrent disclosure are provided in SEQ ID NO: 13204-14121 and SEQ ID NO:14122-15039, respectively (Tables 6A, B) of PCT/US18/53247 and in Tables5-6 in PCT/US2017/064379, which are incorporated herein by reference.

“Chimeric antigen receptors” (CARs) are artificial (non-naturallyoccurring) immune cell (e.g., T cell) receptors contemplated for use asa therapy for cancer, using a technique called adoptive cell transfer.

The term “SABR” or “Synthetic Antigen Binding Receptor”, “SyntheticAntigen Receptor (SAR)” or “Antigen binding Receptor” or “ABR” asdescribed herein refers to any receptor that has an antigen bindingdomain and is capable of transmitting a signal to an immune cell (e.g.,T cell, NK cell, macrophage etc.) when expressed in the said immunecell. SABR when expressed in an effector cell, provides the cell withspecificity for a target cell (e.g., a virally infected cell or a cancercell) that express the antigen bound by the SABR. The antigen bindingdomain of a SABR may comprise of a scFv, a vL, vH, antibody, antibodyfragment (e.g., Fab), antibody like moiety, Vα, Vβ, cytokine, receptoretc. in one embodiment, a SABR has at least one antigen binding domainand at least one transmembrane or membrane anchoring domain that allowsit to be expressed on the cell surface. The term “Synthetic AntigenBinding Receptor (SABR)”, as used herein, comprises CARs and alsoencompasses newer approaches to conferring antigen specificity ontocells, such as Antibody-TCR chimeric molecules or Ab-TCR (WO 2017/070608A1 incorporated herein by reference), TCR receptor fusion proteins orTFP (WO 2016/187349 A1 incorporated herein by reference), SyntheticImmune Receptors (SIRs) (see, WO 2018/102795 A1, incorporated herein byreference), Tri-functional T cell antigen coupler (Tri-TAC or TAC) (see,WO 2015/117229 A1, incorporated herein by reference) and zSIR (see,PCT/US2019/035096, incorporated herein by reference). The nucleic acidsequences of several exemplary TFPs comprising the different antigenbinding domains (e.g., vL and vH fragments, vHH, ligands and receptorsetc.) and based on CD3ε, CD3δ, CD3γ and CD3ζ chains and co-expressingthe optional accessory module NEMO-K277A are presented in SEQ IDNO:1900-2205, 2206-2511, 2512-2817, 2818-3123, respectively (Table 13)of PCT/US18/53247, which is incorporated in its entirety by referenceherein. The order of the antigen binding domains contained in theconstruct of different CAR architectures and BiTE listed in Table 13 ofPCT/US18/53247, which is incorporated in its entirety by referenceherein is the same as the order of the constructs on the zCAR-K277Aarchitecture presented in Table 12 of PCT/US18/53247, which isincorporated in its entirety by reference herein. Typically, the term“SABR-T cell” is used, to refer to T-cells that have been engineered toexpress a Synthetic antigen binding receptor. Thus, T lymphocytesbearing such SABRs are generally referred to as SABR-T lymphocytes. Ifthe SABR is a CAR, then the T cells are referred to as CAR-T cells.SABRs can be also expressed in cells other than T cells, such ashematopoietic stem cells, induced pluripotent stem cells (iPSC), NKcells and macrophage.

The term “binding” or “interacting” refers to an association, which maybe a stable association, between two molecules, e.g., between a TCR anda peptide/MHC, due to, for example, electrostatic, hydrophobic, ionicand/or hydrogen-bond interactions under physiological conditions.

The term “single chain variable fragment” or “scFv” refers to an Fvfragment in which the heavy chain domain and the light chain domain arelinked.

The term “Fv fragment” refers to the fragment of an antibody comprisingthe variable domains of its heavy chain and light chain.

The term “specifically bind”, “specific binding”, or “targeting”, asused herein, when referring to a polypeptide (including antibodies) orreceptor, refers to a binding reaction which is determinative of thepresence of the protein or polypeptide or receptor in a heterogeneouspopulation of proteins and other biologicals.

The term “epitope” means a protein determinant capable of specificbinding to an antibody or TCR.

The term “expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed.

“Immune cell” as used herein refers to the cells of the mammalian immunesystem including but not limited to antigen presenting cells, B-cells,basophils, cytotoxic T-cells, dendritic cells, eosinophils,granulocytes, helper T-cells, leukocytes, lymphocytes, macrophages, mastcells, memory cells, monocytes, natural killer cells, neutrophils,phagocytes, plasma cells and T-cells.

“Immune effector cell,” as that term is used herein, refers to a cellthat is involved in an immune response, e.g., in the promotion of animmune effector response.

“Immune effector function” or “immune effector response,” “effectorfunction” refers to the specialized function of a differentiated immunecell.

“Native” or “Naturally occurring” or “endogenous” as used herein refersto a gene, protein, nucleic acid (e.g., DNA, RNA etc.) or fragmentthereof that is native to a cell or is naturally expressed in a cell.

As used herein a “non-naturally occurring agent” or “non-native” or“exogenous” refers to an agent that is not naturally expressed in acell. Stated another way, the non-naturally occurring agent is“engineered” to be expressed in a cell. As used herein a “non-naturallyoccurring immune receptor” or “exogenous immune receptor” refers to animmune receptor that is not naturally expressed in an immune cell.Stated another way, the non-naturally occurring immune receptor is“engineered” to be expressed in an immune cell.

The term “Synthetic Immune Receptor” or alternatively a “SIR” refers tonext generation CAR platforms that are described in WO 2018/102795 A1which is incorporated herein by reference.

As used herein, the phrase “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting an agent from one organ,or portion of the body, to another organ, or portion of the body.

As used herein, “specific binding” refers to the ability of a SAM tobind to a protein or to a peptide presented on an MHC (e.g. class I MECor class II MEC). T

In certain embodiments, agents of the invention may be used alone orconjointly administered with another type of therapeutic agent.

The terms “T-cell” and “T-lymphocyte” are interchangeable and usedsynonymously herein. Examples include but are not limited to naïve Tcells (“lymphocyte progenitors”), central memory T cells, effectormemory T cells, stem memory T cells (Tscm), iPSC-derived T cells,synthetic T cells or combinations thereof.

The term “TCR receptor fusion proteins” or “TFP” refers to a nextgeneration SAR platform as described in WO 2016/187349 A1 which isincorporated herein by reference.

“SARS-cov2” or “novel coronavirus” or “severe acute respiratory syndromecoronavirus 2” as described herein is the etiological agent ofcoronavirus disease 2019 (COVID-19) or a variant thereof.

“SARS-cov2 immune” or “SARS-cov2 positive” as the term used hereinrefers to subjects or donors who have evidence of cellular and/orhumoral immunity to SARS-cov2. In an embodiment, the SARS-cov2 immunesubjects are subjects who have a history of infection with SARS-cov2. Inan embodiment, the SARS-cov2 immune subjects are subjects who have ahistory of having received a SARS-cov2 vaccine. In an embodiment, theSARS-cov2 immune subjects have T cells that recognize one or moreantigens encoded by SARS-cov2. In an embodiment, the SARS-cov2 immunesubjects have antibodies against one or more antigens encoded bySARS-cov2. Exemplary antigens encoded by SARS-cov2 include Spikeglycoprotein, nucleocapsid protein and membrane glycoprotein.

“SARS-Cov2 specific T cells” or “SARS-Cov2 reactive T cells” asdescribed herein refer to T cells that bind to one or more SARS-Cov2peptides when presented by the MHC complex. SARS-Cov2 specific T cellscan be recognized by methods known in the art, such as staining with MHCtetramers.

Matador Assay as the term used herein refers to a non-radioactiveluciferase-based cytotoxicity assay as described in PCT/US2017/024843and Matta H, et al. (2018) Scientific reports 8(1):199, which areincorporated in their entirety by reference herein.

Topanga Assay as the term used herein refers to a non-radioactiveluciferase based assay for detection of an antigen binding domain or anSABR incorporating an antigen binding domain as described inGopalakrishnan R, et al. (2019) Scientific reports 9(1):1957, which isincorporated in their entirety by reference herein.

Malibu-Glo assay as the term used herein refers to a non-radioactiveluciferase based assay for detection of an antigen as described inNatarajan V, et al. (2020) Scientific reports 10(1):2318, which isincorporated in their entirety by reference herein.

Generation and Expansion of Viral Specific (e.g., SARS-Cov2 Specific) TCells for Adoptive Cellular Therapy

In one aspect, the studies disclosed herein seek to determine the impactof various SABR-T cell stimuli amenable to a high-yield manufacturingprocess, and/or for enriching memory T cell immunophenotypes in a finaltherapeutic product. Standard anti-CD3/CD28 bead-based stimulation iswidely used in the field as a method to expand T cells ex vivo or invitro prior to transduction with SABR-encoding vectors. However,disclosed herein are manufacturing processes for SARS-Cov2antigen-stimulated T cells (e.g., SARS-CoV2-specific T cells) that alsoexpress one or more SABRs. Such processes may comprise an initial T-cellenrichment step, wherein CD3⁺ T cells are enriched/purified from a moreheterogeneous mixture of cells (e.g., from whole blood, from PBMCs, andthe like); stimulated to recognize and respond to pre-selected antigens(e.g., viral antigens or other tumor/disease-associated antigens, etc.);and transduced with one or more SABR constructs. For example, a sampleof cells obtained from a SARS-Cov2 subject comprising CD3⁺ T cells(e.g., PBMC) may be enriched for CD3⁺ T cells, and said CD3⁺ T cellsbrought into contact with antigen-presenting stimulator cells.

In an embodiment, the SARS-Cov2 specific T cells are directly used tomanufacture SABR-T cells. In an embodiment, the SARS-Cov2 specific Tcells are used to generate iPSC cells. In an embodiment, the iPSC(induced pluripotent stem cells) generated from SARS-Cov2 specific Tcells are used to express SABR and then differentiated into SARS-Cov2specific T cells expressing the SABR. Methods to generate iPSC from Tcells and methods to generate T cells from iPSC are known in the art.

In an embodiment, the CD3⁺ T cells are isolated from the sample and thenexpanded by making contact with the SARS-Cov2 antigen-presentingstimulator cells. More preferably, the T cells and SARS-Cov2antigen-presenting stimulator cells (e.g., BLCLs, T cells) are derivedfrom the same sample and are thus HLA-matched. Such cell selectionmethods and techniques generally include positive selection of CD3⁺and/or CD19⁺ cells from the sample and/or negative selection bydepletion of undesired cells or components from the sample. For example,and without limitation, such methods comprise selection with live cellsorting techniques (e g, fluorescence activated cell sorting), anti-CD3and/or anti-CD19 beads (e.g., magnetic beads), plastic adherence,depletion of B cells, elutriation, and/or combinations thereof. Aportion of the resultant CD3⁺ T cells may be transduced with a viralvector encoding a synthetic antigen binding receptor or SABR (e.g. aCAR, SIR, Ab-TCR, cTCR, TFP etc.) before and/or after contact withantigen-presenting stimulator cells. In an exemplary embodiment, SABRtargeting CD19 are presented in SEQ ID NO: 253-308 (Table 8). In anembodiment, the remaining CD3⁺ T cell population may be transduced witha vector encoding one or more proteins or peptides encoded by a virus(e.g., SARS-cov2) and expanded to generate antigen-presenting stimulatorcells. In an exemplary embodiment, SARS-cov2 encoded proteins that canbe used for generation of stimulator cells are provided in nucleic acidSEQ ID NO:231-234 and amino acid SEQ ID NO:701-704 (Table 7). SARS-cov2encoded polypeptides and polyepitopes that can be used for generation ofstimulator cells are provided in nucleic acid SEQ ID NO:225-229 andamino acid SEQ ID NO:695-699 (Table 7). In an embodiment, B cells orBLCLs may be transduced with a vector encoding one or more proteins (SEQNO:701-704) or polypeptides (SEQ ID NO:225-229) encoded by a virus(e.g., SARS-cov2) and expanded to generate antigen-presenting stimulatorcells.

Until now, methods for the manufacture of SABR-T cells known in the arthave relied on either a crude and heterogeneous starting material, suchas PBMC (see Sun et al. Journal for ImmunoTherapy of Cancer (2015) 3:5)or highly purified isolates of specific T cell types, i.e., CD4⁺, CD8⁺ Tcells, or specific ratios thereof (see Terakura et al. Blood (2011) 119:1 and Turtle et al. Sci Transl Med. (2016) September 7; 8). However, theinvention disclosed herein provides ex vivo methods for enrichingSARS-cov2 antigen-specific T cells to be used in the manufacture ofSABR-T cells and for use as cellular vaccine. Notably, in some preferredembodiments, such methods comprise obtaining a sample of cells (e.g.,PBMC) from a SARS-cov2-immune subject comprising CD3⁺ cells andcontacting said CD3⁺ cells with SARS-cov2 antigen-presenting stimulatorcells. In an embodiment, the CD3⁺ T cells are isolated from the sampleprior to contacting the SARS-cov2 antigen-presenting stimulator cells bymethods known in the art (e.g., positive selection of CD3⁺ cells fromthe sample and/or negative selection by depletion of undesired cells orcomponents from the sample). For example, and without limitation, suchmethods include selection using fluorescence activated cell sorting(FACS), with anti-CD3 beads (e.g., magnetic beads), plastic adherence,depletion of NK cells using anti-CD56, elutriation, and/or combinationsthereof. Sensitizing the selected CD3⁺ cells to SARS-cov2 viral antigen,may promote a central memory phenotype in the resultant SARS-cov2antigen-specific T cell population. Such cells may be transduced with aviral vector encoding a synthetic antigen binding receptor (SABR) beforeand/or after contact with the SARS-cov2 antigen-presenting stimulatorcells. In an exemplary embodiment, T cells are transduced with one ormore SABRs targeting CD19, which are presented in SEQ ID NO: 253-308(Table 8). Exemplary SABR targeting other antigens are known in the artand described in WO 2017/070608 A1, WO 2016/187349 A1, WO 2018/102795A1, WO 2015/117229 A1, PCT/US2019/035096, which are all incorporatedherein by reference. In some such embodiments the SABR-expressingSARS-cov2 antigen-specific CD3⁺ T cells are cultured with the SARS-cov2antigen-presenting stimulator cells.

The initial CD3⁺ enrichment step disclosed herein provides a startingmaterial that is significantly less heterogeneous than a PBMC sample yetretains some level of heterogeneity over highly purified cell fractions.Without wishing to be bound by theory, it is postulated that by using aninitial CD3⁺ enrichment step, the starting material comprises a mixtureof cells (including at least a plurality of effector T cell types, Thelper cells (CD4⁺ T cells/TH cells), cytotoxic T cells (cD8⁺ Tcells/CTLs), memory T cell types (i.e., central memory T cells (TCMcells), effector memory T cells (TEM cells), tissue resident memory Tcells (TRI), and virtual memory T cells (TVM cells)), regulatory T cells(Treg cells), natural killer T cells (NKT cells), mucosal associatedinvariant cells (MAIT cells), gamma delta T cells (gd T cells),double-negative T cells (DNTs), CD3⁺ B cells, or any combinationthereof) which may work synergistically or provide an advantageousmilieu to promote increased viability and proliferation ofSARS-cov2-antigen-specific T cells following contact withSARS-cov2-presenting APCs, improved transduction efficiency ofSABR-expressing vector in such SARS-cov2 antigen-specific T cells, and ahigher percentage of TCM cells in the final therapeutic composition.

In some embodiments, further enrichment is conducted following T cellstimulation with a SARS-cov2 antigen. For example, NK depletion (e.g.,CD56 depletion) may be employed prior to a subsequent antigenstimulation step (i.e. prior to re-stimulation of enriched, SARS-cov2antigen-specific, T cells with one or more SARS-cov2 antigens)

Stimulation and Transduction

Manufacture of T cells expressing an SABR (e.g., a CAR, SIR, Ab-TCR, orrecombinant TCR) that specifically binds to a protein or a peptidepresented on a class I MHC requires T cell expansion against definedantigens. In some embodiments, the T cells express a SABR (e.g., a CAR,SIR, Ab-TCR, TFP, recombinant TCR etc.) that specifically binds to adisease-associated peptide (e.g., a tumor-associate peptide, antigen,ligand, or the like) or protein antigen (e.g., CD19, CD20, CD22, BCMA,Mesothelin, PSMA etc.).

SARS-cov2 antigen delivery may be via viral infection by native virus,or via transduction using recombinant virus, of a sample of peripheralblood mononuclear cells (PBMCs) from healthy donors, isolated B celllymphoblastoid cells (e.g., CD19⁺ BLEU) therefrom or isolated T cells(e.g., CD3⁺ T cells) therefrom. In an embodiment, SARS-cov2 antigendelivery may be via viral infection by native virus, or via transductionusing recombinant virus, of an antigen presenting cell line. Exemplaryantigen presenting cell lines include, but are not limited to, K562,REC-1, MINO, JEKO-1, and GRANTA-519. In an embodiment, in addition topresenting SARS-cov2 specific antigens, the APC (e.g., K562, REC-1,MINO, JEKO-1, and GRANTA-519 cells) are engineered to express one ormore co-stimulatory molecules (e.g., 4-1BBL, CD32, CD80, CD83 and CD86)or cytokines (IL2, IL15 and IL21 etc.).

Thus, the infected or transduced cells act as antigen-presenting cellsand are referred to as “stimulators”. In certain embodiments, thestimulator cells also express a peptide (i.e., antigen) or protein onthe cell surface that is recognized by the SABR. The stimulator cellsmay endogenously express such SABR-targeted peptides (e.g., peptidesderived from CD1.9, CD20, CD22, NY-ESO-1, WT1, Myc, AFP etc.) orproteins (e.g., CD19, CD20, CD22, BCMA, Mesothelin, PSMA etc.), orprotein fragments (e.g. tCD19, tBCMA etc.) or be engineered to expresssuch peptides/proteins. The SEQ ID of tEGFRviii, tCD19 and tBCMA areprovided in SEQ ID NO: 194-196 (Table 6). The stimulator cells may alsoco-express a protein that selectively activates the NE-KB pathway. Thenucleic acid and amino acid sequence of vFLIP K13, an exemplary proteinthat selectively activates NF-κB pathway, are provided in SEQ ID NO: 199and 669, respectively. Other exemplary proteins known to selectivelyactivate the NF-κB pathway include NEMO-K277A and constitutive activemutants of IKKα, IKKβ, NIK and MYD88. The viral vector used to transducestimulators may be a recombinant, replication incompetent virus (e.g.,an adenovirus or a lentiviral vector such as pLENTI-EF1α; SEQ ID NO: 1).Alternatively, said stimulator cells are infected with a wildtype/native virus.

A separate sample or culture (e.g., PBMCs from the same donor that arenot used for transduction, a sample of PBMCs from a different donor, orCD3⁺ cells isolated therefrom) comprises “responders” and contains Tcells that become the active component of a therapy, expressing a T cellreceptor that specifically binds to a peptide or protein antigenpresented by stimulator cells.

In an embodiment, SARS-Cov2 specific T cells are obtained from anSARS-CoV2 immune subject. In an embodiment, SARS-Cov2 specific T cellsare obtained from a SARS-CoV2 immune subject who has received aSARS-Cov2 specific vaccine. In an embodiment, SARS-Cov2 specific T cellsare obtained from a SARS-CoV2 immune subject who has received a boosterdose of a SARS-Cov2 specific vaccine. In an embodiment, the SARS-Cov2specific T cells are obtained from a subject 1 day, 2 days, 3 days, 4days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days,13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days,21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days,29 days, 30 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or2 years after receiving a primary and/or booster dose of a SARS-Cov2specific vaccine. In an exemplary embodiment, the SARS-CoV2 vaccine is avaccine that is manufactured by Moderna, Pfizer, Astrazaneca or Johnsonand Johnson. The SARS-Cov2 specific T cells can be isolate by any of themethods known in the art.

In an embodiment, the donor from whom the responder T cells arecollected is a SARS-cov2 immune donor who has recovered from a SARS-cov2infection and/or shows evidence of cellular and/or humoral immunity toSARS-cov2. A number of test to determine cellular and humoral immunityto SARS-cov2 are known in the art, including, but not limited, topresence of IgG antibodies or cells specific to one or more proteins(e.g. Spike glycoprotein or nucleocapsid protein) or peptide antigensencoded by SARS-cov2. In an embodiment, the donor from whom theresponder T cells are collected is a SARS-cov2 immune donor who hasreceived a SARS-cov2 vaccine and shows evidence of cellular and/orhumoral immunity to SARS-cov2. In an embodiment, the donor from whom theresponder T cells are collected is a SARS-cov2 immune donor who isadministered one or more booster doses of a SARS-cov2 vaccine prior tocollection of PBMC and T cells. A number of SARS-cov2 vaccines are beingtested and are known in the art. In an embodiment, the donor is given upto 5 or more booster doses (e.g., 2, 3, 4, 5 booster doses etc.) ofSARS-cov2 vaccine prior to collection of PBMC and I cells. In anembodiment, the PBMC and T cells are collected from the donor between 1and 30 days after the last dose of the SARS-cov2 vaccine. In anembodiment, the donor from whom the responder T cells are collected is aSARS-cov2 immune donor who is selected for donation based on the levelof SARS-cov2 specific antibodies (e.g., level of IgG antibodies againstSARS-cov2) or percentage of SARS-cov2 reactive CD4 and CD8 T cells asdetermined by methods known in the art (Grifoni et al., 2020, Cell Host& Microbe 27, 671-680). In an embodiment, the donor is selected based onthe level of SARS-cov2 specific antibodies that are at least 20% higherthan a reference control (i.e., a subject who is not SARS-cov2 immune).In an embodiment, the donor is selected based on the level of SARS-cov2specific T cells (CD4 or CD8 T cells) that are at least 20% higher thana reference control (i.e., a subject who is not SARS-cov2 immune). In anembodiment, the donor is selected based on the level of SARS-cov2specific IgG antibodies that are in the top 20, 30, 40, 50, 60, 70, 80,90 percentile of the SARS-cov2 immune subjects. In an embodiment, thedonor is selected if they show level of SARS-cov2 specific T cells (Cator CD8 T cells) that are at least 20% higher than a reference control(i.e., a subject who is not SARS-cov2 immune). In an embodiment, thedonor is selected if they show level of SARS-cov2 specific T cells (CD4or CD8 T cells) that are in the top 20, 30, 40, 50, 60, 70, 80, 90percentile of the SARS-cov2 immune subjects.

In another embodiment, the donor from whom the responder I cells arecollected is not a SARS-cov2 immune donor.

The responder T cells may be isolated from PBMC by any suitable method,of which many are well known in the art. In an embodiment, the“stimulator” cells (e.g., PBMCs, I cells or BLCLs) are obtained from thesame cell population (e.g., PBMC sample) as are the “responder” cellssuch that they are identically HLA-matched. For example, a PBMC samplefrom a donor is split into a “stimulator” cell fraction and a“responder” cell fraction, wherein the responder cells may be enrichedfor CD3⁺ cells; and stimulator cells are transduced or infected so as topresent particular SARS-cov2 antigens on the cell surface. In analternate embodiment, the PBMC sample from a donor is enriched for CD3⁺cells, which are then split into “stimulator” cell fraction and a“responder” cell fraction. The “stimulator” CD3⁺ cell fraction istransduced or infected so as to present particular SARS-cov2 antigens onthe cell surface. Optionally, the stimulator cell fraction may beenriched by methods known in the art prior to transduction/infection,such as by selection for CD19⁺ cells. Thus, the SARS-cov2antigen-presenting cells (e.g., PBMCs, CD3⁺T cells, or BLCLs) willpresent the SARS-cov2 antigen to responder T cells (e.g., CD3⁺-enrichedcells), thus activating and inducing proliferation of SARS-cov2antigen-specific T cells. In some such embodiments, the responder Tcells (e.g., isolated T cells) are transduced with a SABR-encodingvector prior to presentation of SARS-cov2 antigen by the stimulatorfraction (e.g., PBMCs, CD3⁺T cells, or BLCLs). In certain embodiments,the responder T cells (e.g., isolated. T cells) are transduced with aSABR-encoding vector after presentation of SARS-cov2 antigen by thestimulator fraction (e.g., PBMCs, CD3⁺T cells, or BLCLs). In alternateembodiment, cell lines (e.g., aK562, JEMO-1, REC-1 etc.) can betransduced or infected so as to present particular SARS-cov2 antigens onthe cell surface and act as “stimulator” or antigen presenting cells.

Accordingly, provided herein are methods of generating allogeneic orautologous T cells that express a T cell receptor that specificallybinds to, for example, an SARS-cov2 peptide presented on a class I or IIMHC and further express a synthetic antigen binding receptor (SABR) thatbinds to a selected target (e.g., CD 19). In some embodiments, APCs aregenerated through viral infection of stimulator cells, e.g., by wildtype/native SARS-cov2 or an adenoviral vector that encodes a polyepitopeof defined CTL epitopes from SARS-cov2 Spike Glycoprotein, nucleocapsideprotein, membrane glycoprotein sequence or orf 1 ab. In someembodiments, the stimulator cells are mixed with non-infected, isolatedT cells (responders) so as to present the SARS-cov2 polyepitopes to saidT cells. In some embodiments, isolated, virus-specific T cells presentedwith SARS-cov2 polyepitopes are activated and induced for proliferation.

In some embodiments, the responder cells and the stimulator cells areeach derived from peripheral blood mononuclear cells (PBMC). In somesuch embodiments, the responder cells and the stimulator cells are eachderived from PBMCs from the same donor. In other embodiments, theresponder cells and the stimulator cells are each derived from PBMCsfrom different donors. In other embodiments, the responder cells derivedfrom PBMCs from a SARS-cov2 immune donor, e.g., a SARS-cov2 positivedonor, e.g., a donor with history of infection with SARS-cov2 or ahistory of immunization with a SARS-cov2 vaccine. In some suchembodiments, prior to contact with stimulator cells, responder cells areisolated and/or purified so as to consist essentially of T cells. Inpreferred embodiments, the responder cells are isolated and/or purifiedso as to consist essentially of CD3⁺ cells. Most preferably, theresponder cells consist essentially of CD3⁺ T cells.

Prior to presentation to responder cells (e.g., T cells), stimulatorcells may be infected with a native virus, such as SARS-cov2, therebypresenting viral antigens on their surface. In some embodiments, thestimulator cells are transduced with a viral vector, preferably anadenoviral vector or a lentiviral vector, comprising a nucleic acidsequence encoding a SARS-cov2 antigen. In some such embodiments, theadenoviral vector is replication incompetent. More preferably, thevector comprises a nucleic acid sequence encoding one or more SARS-cov2antigens. The one or more SARS-cov2 antigens may comprise a SpikeGlycoprotein (SEQ II) NO: 231 and SEQ ID NO: 701) or fragment thereof(e.g., S1-RBD), S2 protein (SEQ ID NO: 694) or fragment thereof, anucleocapsid polypeptide (SEQ ID NO: 232 and SEQ ID NO: 702) or fragmentthereof, membrane glycoprotein (SEQ ID NO: 233 and SEQ ID NO: 703) orfragment thereof, orf3 (SEQ ID NO: 234 and 704) or fragment thereofand/or orf 1 ab or fragment thereof. In an embodiment, the SARS-cov2antigen is the receptor binding domain of the Spike glycoprotein(S1-RBD) (SEQ ID NO: 230 and 700). In an embodiment the S1-RBD isexpressed on the cell surface in fusion with a heterologoustransmembrane domain or a membrane anchoring domain. The nucleic acidand amino acid sequence of an exemplary S1-RBD fusion protein comprisingthe S1-RBD in fusion with the hinge and transmembrane domains of mouseCD8 are presented in SEQ ID NO:229 and 699, respectively.

In an embodiment, the one or more SARS-cov2 antigens are expressed infusion with a protein degradation domain, such as ubiquitin or aubiquitin mutant. An exemplary ubiquitin mutant is ubiquitin G67A. Theprotein degradation domain may be fused to the amino or the carboxyterminus of the one or more SARS-cov2 antigens.

In an embodiment, the one or more SARS-cov2 antigen proteins areexpressed in fusion with a drug inducible protein degradation domain(DIPDD). Exemplary DIPDD include FKBP12-F36V (SEQ ID NO: 1161),IKZF1-ZF2-145-167 (SEQ ID NO: 1162), IKZF1-ZF2-ZF3-145-197 (SEQ ID NO:1162) and IKZF1-ZF2-ZF3-145-243 (SEQ ID NO: 1163). The DIPDD can beattached to the N- or the C-terminus of the SARS-cov2 encoded proteinsand/or polyepitopes. Exemplary SARS-cov2 proteins and polyepitopes infusion with the different DIPDD are represented by SEQ ID NO: 1176-1195.The proteins carrying the FKBP12-F36V (SEQ ID NO: 1161) DIPDD aredegraded by addition of dTAG13 and its homologs, while proteins carryingIKZF1-ZF2-145-167 (SEQ ID NO: 1162), IKZF1-ZF2-ZF3-145-197 (SEQ ID NO:1162) and IKZF1-ZF2-ZF3-145-243 (SEQ ID NO: 1163) are degraded by theaddition of IMiDs (immune modulatory drugs), such as pomalidomide,lenalidomide, CC220, and CC885. Other DIPDDs are known in the art andcan be used in alternate embodiment of the disclosure. In an embodiment,the stimulator cells expressing the SARS-cov2 antigen-DIPDD fusions aretreated with dTAG13 or IMIDs in vitro. In an embodiment, the stimulatorcells expressing the SARS-cov2 antigen-DIPDD fusions are treated withdTAG13 or IMIDs in vivo. In an embodiment, treatment with dTAG13 orIMIDs is carried out for between 1-30 days. In an embodiment, cell aretreated with dTAG13 or its homologs at concentration of between 1 nM to1 μM. In an embodiment, cell are treated with IMIDs at concentration ofbetween 10 pM to 1 μM.

In an embodiment, the vector encodes poly epitope derived from differentantigens of SARS-cov2, such as spike glycoprotein, nucleocapsid andmembrane glycoprotein, orf3a and/or orf 1 ab. In an embodiment, thevector encodes a poly epitope of defined CTL epitopes from spikeglycoprotein, nucleocapsid and membrane glycoprotein, orf3a and/or orf 1ab. An exemplary poly epitope derived from SARS-cov2 antigens that arepresented by HLA-A0201 is represented by nucleic acid and amino acid SEQII) NO: 225 and 695, respectively. In an embodiment, the vector encodesa poly epitope of defined cm epitopes from spike glycoprotein,nucleocapsid and membrane glycoprotein and/or orf 1 ab fused toubiquitin or a mutant thereof. An exemplary ubiquitin mutant isubiquitin G67A (SEQ ID NO: 212 and SEQ ID NO: 682). The proteindegradation domain may be fused to the amino or the carboxy, terminus ofthe one or more SARS-cov2 antigens or polyepitopes. An exemplary polyepitope derived from SARS-cov2 antigens that is presented by HLA-A0201and in fusion with a Ub-G67A mutant is represented by nucleic acid andamino acid SEQ ID NO: 226 and 696, respectively. An exemplary polyepitope derived from SARS-cov2 antigens that is presented byHLA-DRB10401 is represented by nucleic acid and amino acid SEQ ID NO:227 and 697, respectively. An exemplary poly epitope derived fromSARS-cov2 antigens that is presented by HLA-DRB10401 and in fusion witha Ub-G67A mutant is represented by nucleic acid and amino acid SEQ IDNO: 228 and 698, respectively. In an embodiment, the vector encodes apoly epitope derived from spike glycoprotein, nucleocapsid and membraneglycoprotein and/or orf 1 ab fused to one or more proteins encoded bySARS-cov2. In an embodiment, the vector encodes a poly epitope ofdefined CTL epitopes from nucleocapsid and membrane glycoprotein fusedto Spike Glycoprotein.

In some embodiments, the stimulator cells are incubated with one or morecytokines prior to culturing with (i.e., presentation to) respondercells (e.g., non-infected PBMC or CD3⁺ enriched cells). Such stimulatorcells may comprise B cells (e.g., BLCLs), antigen-presenting T-cells,dendritic cells, artificial antigen-presenting cells, and/or aK562cells. In preferred embodiments, the stimulator cells areantigen-presenting BLCLs. In some embodiments, the stimulator cells alsoexpress a module that results in NF-κB activation. Exemplary module thatresults in NF-κB activation is vFLIP K13 (SEQ ID NO: 199 and SEQ ID NO:669) and hNEMO-K277A.

Though antigen-specific cells achieve activation and proliferation whenpresented with antigen by the stimulator fraction, such stimulator cellsare not desirable in the final harvested SABR-T cell product. Moreover,in order to minimize the risk of any viral recombination events inproliferating cells leading to formation of competent virus, thestimulator cells are treated and/or modified prior to culturing withresponder T cells so as to inhibit proliferation, e.g., by irradiationwith gamma rays or exposure to an agent such as mitomycin C. Forexample, in such culture conditions, responder cells (e.g., I-cells) arepresented with peptide antigens by non-proliferating stimulator cells.In some such embodiments, the culture is maintained from at least 24hours to at least 28 days prior to transduction with a SABR-encodingvector. In some embodiments, the culture is maintained for at least 24hours, at least 2 days, at least 3 days, at least 4 days, at least 0.5days, at least 6 days, at least 7 days, at least 8 days, at least 9days, at least 9 days, at least 10 days, at least 11 days, at least 12days, at least 13 days, at least 14 days, at least 17 days, or at least28 days prior to transduction with a SABR-encoding vector. Preferably,the culture is maintained for at least 2 days after antigen presentationby stimulator cells prior to transduction with a SABR-encoding vector,Most preferably, the culture is maintained for at least 6 days afterantigen presentation by stimulator cells prior to transduction with aSABR-encoding vector. In further embodiments, the culture is maintainedfrom at least 24 hours to at least 28 days following transduction with aSABR-encoding vector. In certain embodiments, the culture is maintainedfor at least 24 hours, at least 2 days, at least 3 days, at least 4days, at least 5 days, at least 6 days, at least 7 days, at least 8days, at least 9 days, at least 9 days, at least 10 days, at least 11days, at least 12 days, at least 13 days, at least 14 days, at least 17days, or at least 28 days following transduction with a SABR-encodingvector. In some embodiments, the culture is re-seeded and/orre-stimulated as necessary.

For example, responder T cells undergo at least a first stimulation step(i.e., presented with antigen on APCs) and may be re-seeded and/orre-stimulated as needed (i.e., a second time or more). Said re-seededand/or re-stimulated culture is maintained for at least 24 hours, atleast 3 days, at least 9 days, at least 11 days, at least 14 days, atleast 17 days, or at least 28 days prior to/or following transductionwith a SABR-encoding vector. In preferred embodiments, a responder cellculture is stimulated at least once prior to transduction with aSABR-encoding vector. More preferably, the responder culture isstimulated multiple times, wherein subsequent stimulation steps areseparated by anywhere from 2 to 14 days. For example, a cultureundergoing a first stimulation may undergo a second stimulation (e.g.,re-stimulation) 11 days after the first stimulation; optionally, a thirdstimulation (e.g., re-stimulation) is initiated 7 days after the secondstimulation step. A culture undergoing a stimulation step (e.g.,re-stimulation) is maintained for at least 1 to 10 days beforetransduction with a SABR-encoding vector. Preferably, the culture ismaintained for at least 2 days before transduction with a SABR-encodingvector. Most preferably, the culture is maintained for at least 6 daysafter antigen presentation by stimulator cells prior to transductionwith a SABR-encoding vector. Optionally, an NK depletion step isemployed prior to stimulation. For Example, CD56⁺ cells may be depletedfrom culture (e.g. with anti-CD56 beads) immediately prior tostimulation by APCs.

In certain embodiments, after at least a first presentation of antigento achieve activation and proliferation, the SARS-cov2 antigen-specificT-cells may be frozen and stored prior to and/or following transductionwith a SABR-encoding vector, to be thawed at a future date. In someembodiments, said thawed culture is re-stimulated and/or re seeded asnecessary, and said re-stimulated and/or re-seeded culture is maintainedfor at least 24 hours, at least 2 days, at least 3 days, at least 9days, at least 11 days, at least 14 days, at least 17 days, or at least28 days prior to/or following transduction with a SABR-encoding vectoras described herein. Likewise, in such cases wherein the culture is restimulated multiple times, subsequent stimulation steps are separated byanywhere from 2 to 14 days, as described herein.

The activation and proliferation of antigen-specific T cells alsorequires a sufficient amount of antigen-presentation by stimulatorcells. Accordingly, the stimulation cultures contemplated herein (e.g.,including re-stimulation cultures) comprise known ratios of respondercells to simulator cells. For example, the ratio of responder cells tosimulator cells is about 0.1:1 to about 20:1. For example, and withoutlimitation, in preferred embodiments, the initial stimulation comprisesa responder: stimulator ratio of about 0.43:1. A subsequent stimulation(e.g., re stimulation) may comprise a responder: stimulator ratio of0.25:1 and a yet further stimulation (e.g., re-stimulation) comprises aresponder: stimulator ratio of 4:1.

In certain aspects, provided herein are methods of generating allogeneicor autologous SABR-T cells expressing TCRs that specifically bind topeptides (e.g., antigens) comprising T cell epitopes presented on MHC(e.g. class I MHC), and SABRs that bind to a selected target, such as adisease-associated peptide target, for treating autoimmune disorders andcancers (e.g., MS, SAD, IBD, and/or CD19⁺ B-cell malignancies,lymphomas, leukemias, and/or solid tumors). Contemplated herein are Tcells suitable for the production of SABR-T cells generated byincubating a sample comprising T cells (e.g., a PBMC sample or CD3⁺cells isolated therefrom) with antigen-presenting cells (APCs) thatpresent one or more of the T cell epitopes described herein (e.g., APCsthat present a peptide described herein comprising a SARS-cov2 epitopeon a MHC complex, such as SARS-cov2-infected or recombinantly transducedBLCLs, T cells or aK562).

In some embodiments, the peptides comprising a T cell epitope, asdescribed herein, comprise epitope from SARS-cov2. In some embodiments,the epitopes are HLA class restricted T cell epitopes. In otherembodiments, the epitopes are HLA class H-restricted.

The peptides provided herein may comprise a sequence of any SARS-cov2viral protein (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids of any SARS-cov2protein). In some embodiments, the peptides provided herein comprise nomore than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 contiguousamino acids of the SARS-cov2 viral protein.

The peptides provided herein may comprise a sequence of SpikeGlycoprotein (e.g., a sequence of at least 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 contiguous amino acids of SpikeGlycoprotein). In some embodiments, the peptides provided hereincomprise no more than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10contiguous amino acids of Spike Glycoprotein. Several variants of Spikeglycoprotein have been isolated and are covered in alternate embodimentof the disclosure. An exemplary Spike Glycoprotein amino acid sequenceis provided in SEQ ID NO: 701.

In some embodiments, the peptides provided herein comprise a sequence ofSARS-cov2 nucleocapsid phosphoprotein (e.g., a sequence of at least 5,6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19 or 20 contiguous aminoacids). In some embodiments, the peptides provided herein comprise nomore than 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 contiguousamino acids of SARS-cov2 nucleocapsid phosphoprotein. An exemplarySARS-cov2 nucleocapsid phosphoprotein amino acid sequence is provided inSEQ ID NO: 702.

In some embodiments, the peptides provided herein comprise a sequence ofSARS-cov2 membrane glycoprotein (e.g., a sequence of at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous aminoacids of SARS-cov2 membrane glycoprotein). In some embodiments, thepeptides provided herein comprise no more than 25, 20, 19, 18, 17, 16,15, 14, 13, 12, 11 or 10 contiguous amino acids of SARS-cov2 membraneglycoprotein. An exemplary SARS-cov2 membrane glycoprotein amino acidsequence is provided in SEQ ID NO: 703.

Preferably, the peptide comprises the sequence of an epitope listed inTable 13,

TABLE 13 MHC RESTRICTED SARS-cov2 PEPTIDEs SEQ ID MHC NAME NO SEQUENCErestriction SARS-cov2-S-Prt-Pepl 980 KLPDDFTGCV HLA-A*0201SARS-cov2-S-Prt-Pep2 981 ILPDPSKPS HLA-A*0201 SARS-cov2-S-Prt-Pep3 982VLNDILSRL HLA-A*0201 SARS-cov2-S-Prt-Pep4 983 VVFLHVTYV HLA-A2SARS-cov2-S-Prt-Pep5 984 LITGRLQSL HLA-A2 SARS-cov2-S-Prt-Pep6 985FIAGLIAIV HLA-A2 SARS-cov2-S-Prt-Pep7 986 RLNEVAKNL HLA-A*0201SARS-cov2-S-Prt-Pep8 987 NLNESLIDL HLA-A*0201 SARS-cov2-S-Prt-Pep9 988ALNTLVKQL HLA-A*0201 SARS-cov2-S-Prt-Pep10 989 SIIAYTMSL HLA-A*0201SARS-cov2-S-Prt-Pep11 990 IRGWIFGTTLDSKTQSLL DRB1*04:01SARS-cov2-S-Prt-Pep12 991 CTFEYVSQPFLMD DRB1*04:01 SARS-cov2-S-Prt-Pep13992 QPFLMDLEGKQGN DRB1*04:01 SARS-cov2-S-Prt-Pep14 993 TRFQTLLALHRSYLTPGDRB1*04:01 DSSSGW SARS-cov2-S-Prt-Pep15 994 KSFTVEKGIYQTSNFRV DRB1*04:01Q SARS-cov2-S-Prt-Pep16 995 KSFTVEKGIYQTSNFRV DRB1*07:01 QSARS-cov2-S-Prt-Pep17 996 SASFSTFKCYGVSPTKL DRB1*07:01SARS-cov2-S-Prt-Pep18 997 SASFSTFKCYGVSPTKL DR8 SARS-cov2-S-Prt-Pep19998 QLIRAAEIRASANLAATK DRB1*04:01 SARS-cov2-S-Prt-Pep20 999HWFVTQRNFYEPQII DRB1*04:01 SARS-cov2-S-Prt-Pep21 1000 NLDSKVGGNYNYLYRLClass II FR SARS-cov2-S-Prt-Pep22 1001 LLNKHIDAYKTFP HLADR2, DR3SARS-cov2-S-Prt-Pep23 1002 LQLPQGTTL HLA-A*0201SARS-cov2-Nucleocapsid-Pep1 1003 ALNTPKDHI HLA-A*0201SARS-cov2-Nucleocapsid-Pep2 1004 GDAALALLL HLA-B*4001SARS-cov2-Nucleocapsid-Pep3 1005 LLLDRLNQL HLA-A*0201SARS-cov2-Nucleocapsid-Pep4 1006 RLNQLESKM HLA-A*0201SARS-cov2-Nucleocapsid-Pep5 1007 GMSRIGMEV HLA-A*0201SARS-cov2-Nucleocapsid-Pep6 1008 LALLLLDRL HLA-A*0201SARS-cov2-Nucleocapsid-Pep7 1009 GDAALALLLL HLA-A*0201SARS-cov2-Nucleocapsid-Pep8 1010 LLLLDRLNQL HLA-A*0201SARS-cov2-Nucleocapsid-Pep9 1011 TKAYNVTQAF HLA-B*1525SARS-cov2-Nucleocapsid-Pep10 1012 NFKDQVILL HLA-A*2402SARS-cov2-Nucleocapsid-Pep11 1013 KTFPPTEPK HLA-A*1101SARS-cov2-Nucleocapsid-Pep12 1014 MEVTPSGTWL HLA-B*4001SARS-cov2-Nucleocapsid-Pep13 1015 TKAYNVTQAF Class ISARS-cov2-Nucleocapsid-Pep14 1016 NFKDQVILL A*24:02SARS-cov2-Membrane-Glycoprt- 1017 TLACFVLAAV HLA-A*0201 PeplSARS-cov2-Membrane-Glycoprt- 1018 GLMWLSYFI HLA-A*0201 Pep2SARS-cov2-Membrane-Glycoprt- 1019 HLRIAGHHL HLA-B*1502 Pep3SARS-cov2-Membrane-orf1ab- 1020 CLEASFNYL HLA-A*0201 peplSARS-cov2-Membrane-orf1ab- 1021 WLMWLIINL HLA-A*0201 pep2SARS-cov2-Membrane-orf1ab- 1022 ILLLDQALV HLA-A*0201 pep3SARS-cov2-Membrane-orf1ab- 1023 SACVLAAEC HLA-A*0201 pep4SARS-cov2-Membrane-orf1ab- 1024 SLPGVFCGV HLA-A*0201 pep5SARS-cov2-Membrane-orf1b- 1025 TLMNVLTLV HLA-A*0201 pep6SARS-cov2-Membrane-orf1ab- 1026 SMWALIISV HLA-A*0201 pep7SARS-cov2-Membrane-orf3a-pep1 1027 RIFTIGTVTLKQGEI B*58:01SARS-cov2-Membrane-orf3a-pep1 1028 TVTLKQGEI B*58:01

In some embodiments, the peptides provided herein comprise two or moreof the T cell epitopes (e.g., viral epitopes). In some embodiments, thepeptides provided herein comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 T cell epitopes. For example,in some embodiments, the peptides provided herein comprise two or moreof the T cell epitopes connected by linkers (e.g., polypeptide linkers).In some embodiments, the polypeptide or protein further comprises anintervening amino acid sequence between at least two of the plurality ofepitopes. In some embodiments, the intervening amino acids or amino acidsequences are proteasome liberation amino acids or amino acid sequences.Non-limiting examples of proteasome liberation amino acids or amino acidsequences are or comprise AD, K or R. In some embodiments, theintervening amino acids or amino acid sequence are TAP recognitionmotifs. Typically, TAP recognition motifs may conform to the followingformula: (R/N:I/Q:W/Y)n where n is any integer >1. Non-limiting examplesof TAP recognition motifs include RIW, RQW, MIW and NQY, In someembodiments, the epitopes provided herein are linked or joined by theproteasome liberation amino acid sequence and, optionally, the TAPrecognition motif at the carboxyl terminus of each epitope. ExemplarySARS-cov2 polyepitopes containing proteasome liberation sequences andTAP recognition motifs are presented in SEQ NO: 695 and SEQ ID NO:697.

In an embodiment, the one or more SARS-cov2 polyepitopes are expressedin fusion with a drug inducible protein degradation domain (DIPDD).

In some embodiments, the sequence of the peptides comprises a SARS-cov2viral protein sequence except for 1 or more (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) conservative sequence modifications.

In some embodiments, the peptides provided herein comprise a sequencethat is at least 80%, 85%, 90%, 95% or 100% identical to a proteinsequence (e.g., the sequence of a fragment of a viral protein).

The peptide can be a chimeric or fusion peptide. Exemplary SARS-covepolyepitopes containing proteasome liberation sequences and TAPrecognition motifs are presented in SEQ ID NO: 695 and SEQ ID NO:697. Insome embodiments, the peptide provided herein is linked to anotherpeptide comprising a distinct epitope.

In certain aspects, provided herein are nucleic acid molecules encodingthe peptides described herein. In some embodiments, the nucleic acidmolecule is a vector. In some embodiments, the vector provided hereinencodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 epitopes provided herein (e.g., epitopes provided in Table13). In some embodiments, the vector is a viral vector (e.g. alentiviral vector).

In some embodiments, provided herein are cells that contain a nucleicacid described herein (e.g., a nucleic acid encoding a peptide describedherein).

SABR-T Cells

Provided herein are methods of treating cancers, autoimmune diseases(e.g., MS, SAD, IBD) and infectious diseases (e.g., HIV-1, SARS, MERS,SARS-cov2, influenza, EBV, CMV etc.) by administering to the subjectallogeneic or autologous SABR-T cells expressing a T cell receptor thatspecifically binds to an antigen (e.g., a virus peptide antigen)presented on a or major histocompatibility complex (MHC) molecule, and asynthetic antigen binding receptor molecule with the ability to bothspecifically bind a selected target peptide (e.g., a WT1 peptide, or Raspeptide, or NY-ESO peptide) or protein (e.g., CD19, CD20, CD22, CD33,CD123, Mesothelin, BCMA, CD70, Her2, PSMA, CD38, CD138, MPL etc.) andtransduce subsequent activation signals.

In some embodiments, the antigen-specific T cells used for generatingthe SABR-T cells of the invention are selected from a cell bank orlibrary. Preferably, the MHC is a class I MHC. In certain embodiments,the MHC is a class II MHC and has an a (or α) chain polypeptide that isHLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA or HLA-DRA. In some such embodiments,the class II MHC has a b (or β) chain polypeptide that is HLA-DMB,HLA-DOB, HLA-DPB, HLA-DQB or HLA-DRB. Such I cells, as described herein(e.g., antigen-specific T cells and antigen-specific T cells expressinga SABR), are stored in a cell library or bank before they areadministered to the subject.

Also provided herein are APCs that present a peptide described herein(e.g. a peptide comprising a T cell epitope). In some embodiments theAPCs are B cells, antigen presenting T-cells, dendritic cells, orartificial antigen-presenting cells (e.g., aK562

In certain preferred embodiments, the APCs are derived fromlymphoblastoid cells, such as BLCLs. Exemplary antigen-presenting BLCLsare described in O'Reily et al., Immunol Res 2007 38:237-250 and Koehneet al., Blood 2002 99: 1730-1740, which are hereby incorporated byreference. Dendritic cells for use in the process may be prepared bytaking PBMCs from a patient sample and adhering them to plastic. In someembodiments, the APC is an artificial antigen-presenting cell, such asan aK562 cell.

In certain aspects, provided herein are methods of generating APCs thatpresent the one or more of the T cell epitopes described hereincomprising contacting an APC with a SARS-cov2 peptide comprising anepitope and/or with a nucleic acid encoding said epitope. In anembodiment, the SARS-cov2 proteins and/or peptides are presented to APCas fusion with an amphiphile. In some embodiments, the APCs areirradiated. A cell presenting a peptide described herein can be producedby standard techniques known in the art.

In some embodiments, provided herein are T cells (e.g., CD4 T cellsand/or CD8 T cells) that express a TCR (e.g., an αβ TCR or γδ TCR) thatrecognizes a SARS-cov2 peptide described herein presented on a MHC. Insome embodiments, the T cell is a CD8 T cell (a CTL) that expresses aTCR that recognizes a SARS-cov2 peptide described herein presented on aclass I MHC. In some embodiments, the T cell is a CD4 T cell (a helper Tcell) that recognizes a SARS-cov2 peptide described herein presented ona class II MHC.

In some embodiments, provided herein are methods of generating,activating and/or inducing proliferation of SABR-T cells that recognizeone or more of the SARS-cov2 epitopes described herein and also expressa synthetic antigen binding receptor (e.g., SARS-cov2-specific,antiCD19-SABR-T cells). In some embodiments, a culture (or samplethereof) comprising T cells (i.e., isolated T cells) is incubated inculture with an APC provided herein (e.g., an APC that presents apeptide comprising an SARS-cov2 epitope on a class I MHC complex, suchas the virally transduced PBMCs described herein). In some embodiments,the APCs are autologous to the subject from whom the T cells wereobtained. In some embodiments, the APCs are not autologous to thesubject from whom the T cells were obtained. In some embodiments, thesample containing cells are incubated 2 or more times with APCs providedherein. In some embodiments, the T cells (and/or SABR-T cells) areincubated with the APCs in the presence of at least one cytokine. Insome embodiments, the cytokine is IL-2, IL-4, IL-7 and/or IL-15.Similarly, T cell and/or SABR-T cell cultures may be maintained in thepresence at least one cytokine, such as IL-2, IL-7 and/or IL-15.Exemplary methods for inducing proliferation of I cells using APCs areprovided, for example, in U.S. Pat. Pub. No. 2015/0017723, which ishereby incorporated by reference. In preferred embodiments, suchantigen-specific (e.g., SARS-cov2-specific) T cells are transduced witha vector comprising nucleic acid sequence encoding a synthetic antigenbinding receptor (SABR).

Exemplary antigens targeted by SABR include, but are not limited to,tumor antigens, i.e., proteins that are produced by tumor cells thatelicit an immune response, particularly T-cell mediated immuneresponses. The additional antigen binding domain can be an antibody or anatural ligand of the tumor antigen. The selection of the additionalantigen binding domain will depend on the particular type of cancer tobe treated. Tumor antigens are well known in the art and include, forexample, Non-limiting examples of target antigens include: CD5; CD19;CD123; CD22; CD30; CD171; CS1 (also referred to as CD2 subset 1, CRACC,MPL, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 orCLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii);ganglioside G2 (GD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms Like Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; aglycosylated CD43 epitope expressed on acute leukemia or lymphoma butnot on hematopoietic progenitors, a glycosylated CD43 epitope expressedon non-hematopoietic cancers, Carcinoembryonic antigen (CEA); Epithelialcell adhesion molecule (EPCAM); 137113 (CD276); KIT (CD117);Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2);Mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cellantigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascularendothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24;Platelet-derived growth factor receptor beta (PDGFR-beta);Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha(FRa or FR1); Folate receptor beta (FRb); Receptor tyrosine-proteinkinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1);epidermal growth factor receptor (EGFR); neural cell adhesion molecule(NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2mutated (ELF2M); Ephrin 132; fibroblast activation protein alpha (FAP);insulin-like growth factor 1 receptor (IGF-1 receptor), carbonicanhydrase IX (CAlX); Proteasome (Prosome, Macropain) Subunit, Beta Type,9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consistingof breakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1/CD248); tumorendothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroidstimulating hormone receptor (TSHR); G protein coupled receptor class Cgroup 5, member D (GPRC5D); chromosome X open reading frame 61(CXORF61); CD97; CM79a; anaplastic lymphoma kinase (ALK); Polysialicacid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoHglycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1);uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1);adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupledreceptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K);Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading FrameProtein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1(NY-ESO-1); Cancer/testis antigen 2 (LADE-1a); Melanoma-associatedantigen 1 (MAGE-A1); ETS translocation-variant gene 6, located onchromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family,Member 1A (XAGEl); angiopoietin-binding cell surface receptor 2 (Tie 2);melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testisantigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53);p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumorantigen-1 (PCT A-1 or Galectin 8), melanoma antigen recognized by Tcells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerasereverse transcriptase (hTERT); sarcoma translocation breakpoints;melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease,serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V(NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bi;v-myc avian myelocytomatosis viral oncogene neuroblastoma derivedhomolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-relatedprotein 2 (TRP-2); Cytochrome P450 lB 1 (CYPlB 1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS or Brother of the Regulator ofImprinted Sites), Squamous Cell Carcinoma Antigen Recognized. By T Cells3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding proteinsp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); Akinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2(SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renalubiquitous 1 (RUE); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinalcarboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a;CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A), bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); andimmunoglobulin lambda-like polypeptide 1 (IGLL1); MPL, Biotin, c-MYCepitope Tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6, NYBR1, CDH19,CD200R, Slea (CA19.9; Sialyl Lewis Antigen); Fucosyl-GM1, PTK7, gpNMB,CDH1-CD324, DLL3, CD276/B7H3, IL11Ra, IL13Ra2, CD179b-IGLl1,TCRgamma-delta, NKG2D, CD32 (FCGR2A), Tn ag, Tim1-/HVCR1, CSF2RA(GM-CSFR-alpha), TGFbetaR2, Lews Ag, TCR-beta1 chain, TCR-beta2 chain,TCR-gamma chain, TCR-delta chain, FITC, Leutenizing hormone receptor(LHR), Follicle stimulating hormone receptor (FSHR), GonadotropinHormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelopeglycoprotein, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza Ahemagglutinin (HA), GAD, PDL1, Guanylyl cyclase C (GCC), auto antibodyto desmoglein 3 (Dsg3), auto antibody to desmoglein 1 (Dsg1), HLA,HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ,HLA-DR, HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1 (TF1), AFP, GPRC5D,Claudin18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, Liv1,Nectin-4, Cripto, gpA33, BST1/CD157, low conductance chloride channel,the antigen recognized by TNT antibody, CD229, Toso, BAFF-R, SARS-cov2Spike glycoprotein and any combination thereof.

Also disclosed are polynucleotides and polynucleotide vectors encodingthe disclosed target-specific SABRs that allow expression of said SABRsin the disclosed immune effector cells (e.g., T cells, e.g., SARS-cov2specific T cells).

Nucleic acid sequences encoding the disclosed SABRs, and regionsthereof, can be obtained using recombinant methods known in the art,such as, for example by screening libraries from cells expressing thegene, by deriving the gene from a vector known to include the same, orby isolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

Expression of nucleic acids encoding SABRs is typically achieved byoperably linking a nucleic acid encoding the SABR polypeptide to apromoter, and incorporating the construct into an expression vector. Thedisclosed nucleic acid can be cloned into a number of types of vectors.

In another aspect, the disclosure provides an isolated SABR polypeptidemolecule comprising one or more antigen binding domains (e.g., antibodyor antibody fragment, a ligand or a receptor) that bind to antigens asdescribed herein, and are jointed to one or more T cell receptorconstant chains.

In order to assess the expression of a SABR polypeptide or portionsthereof, the expression vector to be introduced into a cell (e.g., anantigen-specific T cell) can also contain either a selectable markergene or a reporter gene or both to facilitate identification andselection of expressing cells from the population of cells sought to betransfected or infected through viral vectors. In some embodiments, theSABR-encoding vector comprises a nucleic acid sequence encoding anantibiotic-resistance gene such as blasticidin resistance (Blast) orpuromycin resistance (PAC). In some such embodiments, the SABR T cells(e.g., the antigen-specific SABR T cells) are cultured in the presenceof the selectable marker (e.g., blasticidin or puromycin), therebyallowing for selection and expansion of SABR-expressing T cellsdescribed herein.

Also provided herein are genetically engineered cells (such as T cells,NK cells, iPSC) comprising vectors encoding nucleic acids encoding SABR(including functional variants). In one embodiment, the cell is selectedfrom the group consisting of a T cell, a Natural Killer (NK) cell, acytotoxic T lymphocyte (CTL), a regulatory T cell, hematopoietic stemcells and/or pluripotent embryonic/induced stem cells. The cell can bean immune effector cell (e.g., a T cell or a NKT cell, or a combinationthereof) or a stem/progenitor cell (e.g., iPSC) that can give rise to animmune effector cell or a synthetic T cell.

In some embodiments, the cell is an immune cell. Non-limiting examplesof immune cells include T-cells and NK-cells. Further, non-limitingexamples of T-cells include Tregs, CD8+ T cells, and CD4+ T cells. Inone embodiment, the cell is a human T cell. In some embodiments, thecell is a human cell. In some embodiments, the cell is a dog cell.

In one embodiment, the cell is a T cell and the T cell is deficient inone or more of endogenous T cell receptor chains. In some embodiments,the cell in the methods is deficient in constant chains of endogenous Tcell receptor α, β1, β2, pre-TCRα, γ or δ or combination thereof. Insome embodiments, the cell in the methods is deficient in HLA antigens.In some embodiments, the cell in the methods is deficient in β2microglobulin.

In some embodiments, the methods provided herein are directed totreating a cancer, infection (e.g., SARS-cov2) and/or an autoimmunedisorder in a subject by administering to the subject autologous orallogeneic SABR-T cells as provided herein.

The methods of the present disclosure rely, in part, on the principlesof I cell restriction to specific target antigens. For example, andwithout limitation, products and preparations comprising donor-derivedCTLs targeted against antigens expressed by a virus, such as SARS-cov2,can be elicited on targeted antigen-presenting (e.g., stimulator) celllines, including SARS-cov2-transformed B Lymphoblasts (BLCLs) or K562cells expressing SARS-cov2 antigens. With respect to the disclosedvirus-antigen-specific SABR T cell preparations, the target cells and/orrecipients of the SABR-T products made by the methods provided herein,may be autologous or allogeneic. In an embodiment, in the case ofallogeneic targets, the human leukocyte antigen (HLA) identity of thedonor must be known and matched to the HLA identity of the target (i.e.,cultured cell lines and/or recipients).

A preparation of SABR T cells may have a mixture of antigen-specific andnon-specific CTLs.

Target cells for the assays disclosed herein are typically selected fortheir uniform phenotype and availability in large quantities. In certainembodiments, the target cell lines are antigen-presenting cells (APCs).In some such embodiments, the target cell lines are B cells,antigen-presenting T-cells, dendritic cells, or artificialantigen-presenting cells (e.g., aK562 cells). In certain embodiments,the target cell lines comprise peripheral blood mononuclear cells(PBMCs). In some such embodiments, the target cell lines arelymphoblasts. In certain preferred embodiments, the target cell linesare virally transformed. In preferred embodiments, the target cell linescomprise each of Phytohemagglutinin-stimulated peripheral bloodlymphocytes (PHA-blasts/PHAbs) and Epstein-Barr virus-transformedB-lymphoblastoid cell lines (BLCLs).

In some embodiments, target cell lines comprise allogeneic cells. Insome such embodiments, target cells carry Human Leukocyte Antigen (HLA)alleles that do not match the HLA alleles to which the SABR-T CTLs ofthe preparation are restricted. In other such embodiments, the targetcells carry HLA alleles that are a partial match to the HLA alleles towhich the SABR-T CTLs of the preparation are restricted. In still othersuch embodiments, the target cells carry HLA alleles that are a match tothe HLA alleles to which the SABR-T CTLs of the preparation arerestricted. In preferred embodiments, the SABR-T CTLs are restricted toHLA alleles of the target cells which encode MHC Class I proteins.

In other embodiments, the target cell lines comprise autologous cells.In some such embodiments the SABR-T CTL preparation is identified assuitable for use against target cells by confirming the ability of thepreparation to lyse two or more autologous target cell lines at, above,or below predetermined thresholds.

In certain embodiments, the autologous or allogeneic SABR-T cellscomprise central memory T cells (TCM e.g., at least 60%, 70%, 80% of thecells are Tcm cells. In some embodiments, the autologous or allogeneicSABR-T cells comprise a ratio of central memory T cells to effectormemory cells (TCM:TEM) from at least 1:1 to at least 3:1, e.g. at least1:1, 1.4:1, 2.5:1, or 3:1). In some such embodiments, the autologous orallogeneic SABR-T cells are predominantly CD4⁺ T cells. In someembodiments, the autologous or allogeneic SABR-T cells comprise at least80% CD4⁺ SABR-T cells and at least 15% CD8⁺ SABR-T cells). In someembodiments, the autologous or allogeneic SABR-T cells comprise a ratioof CD4⁺ T cells to CD8⁺ I cells from at least 1:1 to at least 3:1, e.g.,at least 1:1, 1.4:1, 2.5:1, or 3:1. In some of the methods describedherein, allogenic T cells are selected from a cell bank (e.g., apre-generated third party donor derived bank of epitope-specific Tcells).

In some embodiments, provided herein are methods of treating a cancer ina subject by administering to the subject a therapeutic SABR-T cellpreparation as described herein. In some embodiments, the methodsprovided herein can be used to treat any cancer.

In certain embodiment, the method is used to treat an infectious diseaseor autoimmune disease.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular agent employed, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

In some embodiments, the methods described herein include selectingallogeneic T cells from a cell bank (e.g., a pre-generated third partydonor derived bank of epitope specific T cells). In some embodiments,the T cells are selected because they express a TCR restricted to aclass I MHC that is encoded by an HLA allele that is present in thesubject. In some embodiments, the T cells are selected if the I cellsand subject share at least 2 (e.g., at least 3, at least 4, at least 5,at least 6) HLA alleles and the T cells are restricted through a sharedHLA allele. In some embodiments, the method comprises testing the TCRrepertoire of the pre-generated third-party-donor-derivedepitope-specific T cells (i.e., allogeneic T cells) with flow cytometry.In some embodiments, epitope-specific T cells are detected using atetramer assay, an ELISA assay, a western blot assay, a fluorescentmicroscopy assay, an Edman degradation assay and/or a mass spectrometryassay (e.g., protein sequencing). In some embodiments, the TCRrepertoire is analyzed using a nucleic acid probe, a nucleic acidamplification assay and/or a sequencing assay.

In some embodiments, provided herein are compositions (e.g., therapeuticcompositions) comprising T cells and/or APCs provided herein used totreat and/or prevent an autoimmune disease in a subject by administeringto the subject an effective amount of the composition.

According to the invention, a lymphodepleting treatment is generallyperformed before administering the engineered immune cells to thepatients. Such lymphodepleting treatment generally combines fludarabineand cyclophosphamide. As a preferred embodiment of the presentinvention, the lymphodepleting treatment can comprise an anti-CD52antibody, such as alemtuzumab, alone or in combination. Thelymphodepletion regimen may for instance combine cyclophosphamide,typically for 1 to 3 days, fludarabine for I to 5 days, and alemtuzumabfrom 1 to 5 days. More specifically, the lymphodepletion regimen cancombine between cyclophosphamide 50 and 70 mg/kg/day, fludarabinebetween 20 and 40 mg/m2/day, and alemtuzumab 0.1 to 0.5 mg/kg/day.

According to the present methods, the above induction chemotherapytreatment and lymphodepletion steps are usually followed by a cellimmunotherapy treatment using engineered immune cells.

The immune cells may originate from the patients (autologous engineeredcells) or from donors (allogenic engineered immune cells). They aregenerally primary cells obtainable from leukapheresis or derived fromiPS cells or cell lines. These Immune cells are generally population oflymphocytes, preferably NK or T-cells. The engineered immune cells ofthe present invention preferably express recombinant TCR or a SABRspecific for an antigen marker. By “recombinant TCR” is meant that anexogenous TCR with a different specificity is introduced or expressedinto the cell that partially or completely replace the expression of theendogenous TCR.

In one embodiment, the method includes administering a cell expressingthe SABR molecule, as described herein, in combination with an agentwhich enhances the activity of a SABR-expressing cell, wherein the agentis a cytokine, e.g., IL-2, IL-7, IL-15, IL-21, or a combination thereof.The cytokine can be delivered in combination with, e.g., simultaneouslyor shortly after, administration of the SABR-expressing cell.

In other embodiments, the cells expressing a SABR molecule areadministered in combination with an agent that ameliorates one or moreside effects associated with administration of a cell expressing a SABRmolecule.

SABR Targeting SARS-Cov2

In some embodiment, the disclosure also provides isolated nucleic acidsencoding SABR, wherein the antigen specific domain of the SABR targetsthe spike glycoprotein of SARS-cov2. In an embodiment, the disclosureprovides an isolated nucleic acid encoding SABR, wherein the antigenspecific domain of the SABR targets the Receptor Binding domain of thespike glycoprotein of SARS-cov2. In exemplary embodiments, the nucleicacid sequences of SABR targeting the spike glycoprotein of SARS-cov2 areset forth in SEQ ID NOs: 310-365; 367-422; 424-479 (Tables 9-11).

In some embodiment, the disclosure also provides isolated polypeptidesencoding SABR, wherein the antigen specific domain of the SABR targetsthe spike glycoprotein of SARS-cov2. In an embodiment, the disclosureprovides isolated polypeptides encoding SABR, wherein the antigenspecific domain of the SABR targets the Receptor Binding domain of thespike glycoprotein of SARS-cov2.In exemplary embodiments, thepolypeptide sequences of SABR targeting the spike glycoprotein ofSARS-cov2 are set forth in SEQ ID NOs: 780-835; 837-892; 894-949 (Tables9-11).

The disclosure also provides novel vL, vH and scFv fragments targetingthe spike glycoprotein of SARS-cov2 that can be used in the constructionof single and double chain SABRs, including 2^(nd) generation CARs,single and double chain SIRs, single and double chain cTCRs, Ab-TCR,TFPs, TAC and the like. Exemplary vL, vH and scFv fragments and theirCDRs are listed in Table 7A,

In some embodiments, the antigen specific domains of the SABRs targetingthe Spike glycoprotein comprise one or more V_(L) fragments described inTable 7A. In some embodiments, the polynucleotides encoding the one moreV_(L) fragments comprise, consist of or consist essentially of sequencesset forth in any one or more of SEQ ID NOs: 213 to 215. In someembodiments, the polypeptides encoding the one more V_(L) fragmentscomprise, consist of or consist essentially of sequences set forth inany one or more of SEQ ID NOS: 683 to 685 or sequences with 70-99%identity to sequences set forth in any one or more of SEQ ID NOS: 683 to685 or sequences with 70-99% identity in the three complementaritydetermining regions (CDRs) to the sequences set forth in any one or moreof SEQ ID NOS: 683 to 685 or sequences with less than 3 substitutions inthe three CDRs of the sequences set forth in any one or more of SEQ IDNOS: 683 to 685 or sequences that bind to the same target antigens orthe same epitopes on the target antigens as the sequences set forth inany one or more of SEQ ID NOS: 683 to 685. In some embodiments, theantigen specific domains of the CARs comprise one or more V_(H)fragments described in Table 7a. In some embodiments, thepolynucleotides encoding the one more V_(H) fragments comprise, consistof or consist essentially of sequences set forth in any one or more ofSEQ ID NOS: 217-219. In some embodiments, the polypeptides encoding theone more V_(H) fragments comprise, consist of or consist essentially ofsequences set forth in any one or more of SEQ ID NOS: 687-689 orsequences with 70-99% identity to sequences set forth in any one or moreof SEQ ID NOS: 687-689 or sequences with 70-99% identity in the threecomplementarity determining regions (CDRs) to the sequences set forth inany one or more of SEQ ID NOS: 687-689 or sequences with less than 3substitutions in the three CDRs of the sequences set forth in any one ormore of SEQ ID NOS: 687-689 or sequences that bind to the same targetantigens or the same epitopes on the target antigens as the sequencesset forth in any one or more of SEQ ID NOS: 687-689.

Also provided are functional variants of the SABRs targeting SARS-cov2described herein, which have substantial or significant sequenceidentity or similarity to a parent SABR, which functional variantretains the biological activity of the SABR of which it is a variant.Functional variants encompass, for example, those variants of the SABRdescribed herein (the parent SABR) that retain the ability to recognizeSARS-cov2-infected cells to a similar extent, the same extent, or to ahigher extent, as the parent SABR. In reference to the parent SABR, thefunctional variant can, for instance, be at least about 30%, about 50%,about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%), about 97%, about 98%, about 99%or more identical in amino acid sequence to the parent SABR.

A functional variant can, for example, comprise the amino acid sequenceof the parent SABR with at least one conservative amino acidsubstitution. Alternatively or additionally, the functional variants cancomprise the amino acid sequence of the parent SABR with at least onenon-conservative amino acid substitution. In this case, it is preferablefor the non-conservative amino acid substitution to not interfere withor inhibit the biological activity of the functional variant. Thenon-conservative amino acid substitution may enhance the biologicalactivity of the functional variant, such that the biological activity ofthe functional variant is increased as compared to the parent SABR.

The SABRs (including functional portions and functional variants) can beof any length, i.e., can comprise any number of amino acids, providedthat the SABRs (or functional portions or functional variants thereof)retain their biological activity, e.g., the ability to specifically bindto SARS-cov3 spike glycoprotein, detect SARS-cov2 infected cells in amammal, or treat or prevent SARS-cov2 associated disease (e.g.,COVID-19) in a mammal, etc. For example, the SABR can be about 300 toabout 5000 amino acids long, such as 300, 400, 500, 600, 700, 800, 900,1000 or more amino acids in length.

Further provided herein are vectors encoding nucleic acids encodingSABR, wherein the antigen specific domain of the SABR targets spikeglycoprotein of SARS-cov2.

Also provided herein are genetically engineered cells (such as T cells,NK cells, iPSC) comprising vectors encoding nucleic acids encoding SABR(including functional variants), wherein the antigen specific domain ofthe SABR targets spike glycoprotein of SARS-cov2. In one embodiment, thecell is selected from the group consisting of a T cell, a Natural Killer(NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell,hematopoietic stem cells and/or pluripotent embryonic/induced stemcells. The cell can be an immune effector cell (e.g., a T cell or a NKTcell, or a combination thereof) or a stem/progenitor cell (e.g., iPSC)that can give rise to an immune effector cell or a synthetic T cell.

In some embodiments, the cells are a cell line. Exemplary cell linesthat can be used to express a SABR of the disclosure targeting the spikeglycoprotein of SARS-cov2 include NK92, NK92MI and the like.

In another aspect, the invention relates to a pharmaceutical compositioncomprising a cell or cell population (such as T cells, NK cells, iPSC)comprising vectors encoding nucleic acids encoding SABR, wherein theantigen specific domain of the SABR targets spike glycoprotein ofSARS-cov2.

Cells that express a SABR of the invention are used in the treatment andprevention of SARS-cov2 infection and associated complications (e.g.,COVID-19).

The disclosure provides methods of prevention and treatment ofcomplications (e.g., COVID-19) arising from infection with virusesSARS-cov2 by treatment with a pharmaceutical composition targeting thespike glycoprotein of SARS-cov2 and comprising the cells describedherein, the nucleic acids described herein, the polypeptides encoded bythe nucleic acids described herein or vectors comprising the nucleicacids described herein, and a pharmaceutically acceptable carrier.

The invention thus relates to methods for the prevention and/ortreatment of COVID-19, comprising administering to a subject a cell orcell population comprising a SABR as described herein, said methodcomprising administering, to a subject in need thereof, apharmaceutically active amount of a cell and/or of a pharmaceuticalcomposition of the invention. The invention also relates to a SABR, acell or cell population comprising a SABR as described herein for use intherapy.

In another aspect, the invention relates to a method of providing anSARS-cov2 immunity in a subject, the method comprising administering tothe mammal an effective amount of a cell or cell population geneticallymodified to express a SABR of the invention, wherein the antigen bindingdomain of SABR binds to spike glycoprotein of SARS-cov2.

In another aspect, the invention relates to a method for producing agenetically modified cell or cell population comprising expressing insaid cell or cell population a SABR nucleic acid construct of theinvention. The method may include introducing into the cell a nucleicacid as described herein (e.g., an in vitro transcribed RNA or syntheticRNA; an mRNA sequence encoding a SABR polypeptide as described herein).In embodiments, the RNA expresses the SABR polypeptide transiently. Inone embodiment, the cell is a cell as described herein, e.g., an immuneeffector cell (e.g., T cells or NK cells, or cell population). Cellsproduced by such methods are also within the scope of the invention.

In another aspect, the invention relates to an ex vivo method forgenerating a population of cells for use in adaptive immunotherapycomprising transforming said cell with a SABR of the invention,

Cell Lines for Sars-Cov2 Research and Development and Quality Controland Methods of Using the Same

The disclosure also provides polynucleotides, polypeptides, vectors andprimary cells and cell lines that express the different SARS-cov2antigens and polyepitopes, singly or in combination. Exemplary SARS-cov2antigens include spike glycoprotein and a fragment thereof, nucleocapsidprotein and a fragment thereof membrane glycoprotein and a fragmentthereof. Exemplary cell lines include K562, 293, VeroE6 and the like.

In an embodiment, the primary cells and cell line encodes poly epitopederived from different antigens of SARS-cov2, such as spikeglycoprotein, nucleocapsid and membrane glycoprotein and/or orf 1 ab. Inan embodiment, the SARS-cov2 antigens and polypeptides are expressed inthe primary cells and cell lines stably. In an embodiment, the SARS-cov2antigens and polypeptides are expressed in the primary cells and celllines transiently. In an embodiment, the SARS-cov2 antigens andpolypeptides are expressed in the primary cells and cell lines using aconstitutive promoter (MND promoter, EF1α promoter, CMV promoter etc.).In an embodiment, the SARS-cov2 antigens and polypeptides are expressedin the primary cells and cell lines using an inducible promoter (e.g.,tetracycline promoter). In an embodiment, the SARS-cov2 antigens andpolypeptides are expressed in the primary cells and cell lines followinginfection with SARS-cov2. The disclosure also provides a method for theuse of the primary cells and cell lines as antigen presenting cells(APC) for generation and expansion of SARS-cov2 reactive T cells. Thedisclosure also provides a method for the use of the primary cells andcell lines as antigen presenting cells (APC) for monitoring immuneresponse to SARS-cov2 and for measuring the efficacy of therapeuticstargeting SARS-cov2 (e.g., SARS-cov2 directed SABR, antibodies, antibodydrug conjugates and the like).

In an embodiment, the primary cells and cell lines expressing theSARS-cov2 antigens and polyepitopes, singly or in combination, alsoexpress a luciferase. In an embodiment, the luciferase is a thermostableluciferase. Exemplary thermostable luciferase include marine luciferase(e.g., Gluc, NLuc, PaLuc, TurboLuc and the like) and thermostable beetleluciferase such as (e.g., LucPPE and its engineered variants such asLucPPe-146-1H2, LucPPe-133-1B2, LucPPe-78-0B10, LucPPe49-7C6A andLucPpL-81-6G1. The nucleic acid and amino acid. SEQ ID of exemplaryluciferases are provided in Table 2,

Also provided herein are cell lines and/or compositions comprisingprimary cells and cell lines wherein the cell lines express one or moreof SARS-cov2 antigens, antigen fragments, epitopes, polyepitopes, andalso coexpress the non-secretory forms of GLuc, NLuc, MLuc7, HTLuc,PaLuc1, PaLuc2, MpLuc1, McLuc1, MaLuc1, MoLuc1, MoLuc2, MLuc39, PsLuc1,LoLuc1-3, HtLuc2, TurboLuc16 (TLuc), Renilla Luc, Firefly luciferase(FfLuc or Fluc), LucPPe-146-1H2, LucPPe-133-1B2, LucPPe-78-0B10,LucPPe49-7C6A, LucPpL-81-6G1, CBGRluc, thermostable alkaline phosphataseor homologs or orthologs or mutants or derivatives thereof for thepurpose of use as a target cell line for measuring cytotoxicity.

Also provided herein are primary cells and/or compositions comprisingprimary cells wherein the primary cells express one or more of SARS-cov2antigens, antigen fragments, epitopes, polyepitopes, and also coexpressthe non-secretory forms of GLuc, NLuc, MLuc7, HTLuc, PaLuc 1, PaLuc2,MpLuc1, McLuc1, MaLuc1, MoLuc1, MoLuc2, MLuc39, PsLuc1, LoLuc1-3,HtLuc2, TurboLuc16 (TLuc), Renilla Luc, Firefly luciferase (FfLuc orFluc), LucPPe-146-1H2, LucPPe-133-1B2, LucPPe-78-0B10, LucPPe49-7C6A,LucPpL-81-6G1 or CBGRluc or homologs or orthologs or mutants orderivatives thereof.

In various embodiments, the primary cell and cell lines are modifiedand/or selected to lack the expression of one or more antigens.

The disclosure also provides a method for use of the primary cells andcell lines for measuring the cytotoxicity of SARS-cov2 directedtherapeutics (e.g., I cells, SABR-T, antibodies, bispecific antibodiesetc) using the non-radioactive luciferase based cytotoxicity assay(Matador cytotoxicity assay) as described in PCT/US17/52344, which isincorporated in its entirety by reference herein.

The methods include providing a target cell (e.g., a primary cell orcell line) that has been engineered to express a SARS-cov2 protein(e.g., Spike glycoprotein) or a fragment thereof and is optionallyengineered to express intracellularly a reporter; exposing the targetcell to SARS-cov2 directed agent capable of modulating cytotoxicity; andassaying the cytotoxicity of SARS-cov2 directed agent. In an embodiment,the cytotoxicity of the agent is measured by measuring the activity ofthe reporter. In one embodiment, the reporter is not expressedendogenously by the target cell. In another embodiment, the reporter isexpressed endogenously by the target cell at a level lower than what isachieved by engineered expression. In an embodiment, the reporter is athermostable luciferase. In one embodiment, a change in reporteractivity relative to a reference value is indicative of the agent beingable to modulate cytotoxicity of the target cell. In one embodiment, achange in reporter activity is an increase in reporter activity relativeto a reference value. In some embodiments, the reporter is expressedendogenously by the target cell at a level that is at least 10-25%,25-50%, 50-75% or 75-100% lower than what is achieved by engineeredexpression.

In on embodiment, the reporter activity is assayed in the cell mediacontaining the target cells. In another embodiment, the reporteractivity is assayed in the cell supernatant that is free of the targetcells.

In various embodiments, the cytotoxicity of an agent is measured byobtaining (assaying) the reporter activity in cell pellet and cell freesupernatant and normalizing the reporter activity measured in the cellfree supernatant. In various embodiments, normalizing comprises dividingthe activity in the cell free supernatant by the reporter activitymeasured in the cell pellet

In various embodiments, the reporter is a non-secretory form of anenzyme that is stable under the assay conditions at 37° C. for more than15 min, for more than 30 min, for more than 1 hour, for more than 2hours, for more than 3 hours, for more than 4 hours, for more than 12hours, 24 hours, for more than 36 hours, for more than 48 hours or forat least 96 hours.

In various embodiments, the reporter is a non-secretory form of aluciferase. In some embodiments, the non-secretory form of luciferase isobtained from copepods, deep sea shrimp, beetle, firefly, or homologs ororthologs thereof or mutants or variants or derivatives thereof.

In exemplary embodiments, the copepods are selected from the groupconsisting of any one or more of Gaussia princeps, Pleuromammaabdominalis, Metridia pacifica, Metridia curticauda, Metridiaasymmetrica, Metridia okhotensis, Metridia longa, Lucicutia ovaliformis,Heterorhabdus tanneri, and Pleuromamma scutullata.

In various embodiments, the luciferase is any one or more of GLuc, NLuc,MLuc7, HTLuc, PaLuc1, PaLuc2, MpLuc1, McLuc1, MaLuc1, MoLuc1, MoLuc2,MLuc39, PsLuc1, LoLuc1-3, HtLuc2, TurboLuc16 (TLuc), Lucia Luc, RenillaLuc, Firefly luciferase (FfLuc or Fluc), LucPPe-146-1H2, LucPPe-133-1B2,LucPPe-78-0B10, LucPPe49-7C6A, LucPpL-81-6G1 or CBGRluc or homologs ororthologs or mutants or variants or derivatives thereof.

In various embodiments, the reporter is a non-secretory form of aluciferase obtained from copepods, deep sea shrimp, beetle, firefly orhomologs or orthologs thereof or mutant or derivatives thereof and thereporter activity is assayed by exposing the target cells to aluciferase specific substrate.

In various embodiments, the luciferase-specific substrate iscoelentrazine or an imidazopyrazinone substrate (furimazine) or aderivative thereof. In one embodiment, the luciferase-specific substrateis D-luciferin or a derivative thereof.

In one embodiment, the reporter is a thermostable luciferase. In oneembodiment, the reporter is a thermostable beetle luciferase. In anembodiment, the thermostable beetle luciferase is obtained from Photurispennsylvanica and Pyrophorus plagiothalamus

In some embodiments, the reporter is a non-secretory form of an alkalinephosphatase. In one embodiment, the alkaline phosphatase is aheat-stable alkaline phosphtase.

In some embodiments, the reporter is a non-secretory form of afluorescent protein. In one embodiment, the non-secretory form of afluorescent protein is green fluorescent protein.

In one embodiment, the target cells express a single type of reporter.In another embodiment, the target cells express more than one type ofreporter. In various embodiments, the activity of the two reporters canbe measured independent of each other either simultaneously orsequentially.

In some embodiments, the substrate for one of the reporters iscoelentrazine or an imidazopyrazinone substrate (e.g., furimazine) or aderivative thereof and the substrate for the other reporter isD-luciferin or a derivative thereof.

In some embodiments, the substrate for one of the reporters iscoelentrazine or an imidazopyrazinone substrate (e.g., furimazine) or aderivative thereof and the substrate for the other reporter is pNNP or aderivative thereof.

In some embodiments, the target cells expressing two or more reportersare mixed together prior to the assay.

In some embodiments, the reference value is the reporter activity in anyone or more of (i) target cells that do not express reporter, (ii)target cells that express reporter but are not treated with the testagent(s); (iii) the target cells that are left untreated; (iv) targetcells that are not treated with the substrate for the reporter, or (iii)a combination thereof.

In some embodiments, the agent capable of modulating cytotoxicity is anyone or more of an antibody, small molecule, chemical compound, radiationagent, cytotoxic cells, biologics or combinations thereof.

In some embodiments, the cytotoxic cells are any one or more of T cells,NK cells, PBMCs or combinations thereof. In some embodiments, thecytotoxic cells are modified to express a chimeric or synthetic receptoror a T cell receptor.

In some embodiments, the antibodies are any one or more of chimericantibodies, human antibodies, humanized antibodies, bispecificantibodies, bispecific T cell engager, DART, antibody drug conjugates orcombination thereof.

In some embodiments, the reporter is expressed in cells by any one ormore of plasmid vector, adenoviral vector, adenoassociated viral vector,sleeping beauty transposon, piggyback transposon, pCMV (cytomegalovirus)vectors, vaccinia virus vectors, retroviral vectors, lentiviral vectors,SV40 virus vectors, transfection of naked DNA or transfection of invitro transcribed RNA. In some embodiments, the reporter is expressedusing a non-vector method. In some embodiments, the reporter isexpressed from a foreign promoter. In some embodiments, the reporter isexpressed from a natural promoter.

In some embodiments, the target cells are exposed to the test agent invitro. In some embodiments, the target cells are exposed to the testagent in vivo.

In some embodiments, the assay is performed in vitro. In someembodiments, the assay is performed in a high throughput fashion. Insome embodiments, the assay is performed in vivo. In some embodiments,the target cells are present in a transgenic animal. In someembodiments, the assay is performed to identify agents that increase,decrease or have no effect on the cytotoxicity of an agent on a targetcell. In some embodiments, the assay is performed with one or moreagents used alone or in combination.

In some embodiments, the non-secretory form of the reporter is expressedusing vectors encoding the non-secretory forms of GLuc, NLuc, MLuc7,HTLuc, PaLuc1, PaLuc2, MpLuc1, McLuc1, MaLuc1, MoLuc1, MoLuc2, MLuc39,PsLuc1, LoLuc1-3, HtLuc2, TurboLuc16 (TLuc), Lucia Luc, Renilla Luc,Firefly luciferase (FfLuc or Fluc), LucPPe-146-1H2, LucPPe-133-1B2,LucPPe-78-0B10, LucPPe49-7C6A, LucPpL-81-6G1 or CBGRluc or homologs ororthologs or mutants or derivatives thereof.

In some embodiments, the target cell is a cell line expressing thenon-secretory forms of GLuc, NLuc, MLuc7, HTLuc, PaLuc 1, PaLuc2,MpLuc1, McLuc1, MaLuc1, MoLuc1, MoLuc2, MLuc39, PsLuc1, LoLuc1-3,HtLuc2, TurboLuc16 (TLuc), Renilla Luc, Firefly luciferase (FfLuc orFluc), LucPPe-146-1H2, LucPPe-133-1B2, LucPPe-78-0B10, LucPPe49-7C6A,LucPpL-81-6G1, CBGRluc, thermostable alkaline phosphatase or homologs ororthologs or mutants or derivatives or variant thereof.

In some embodiments, the target cell is a primary cell expressing thenon-secretory forms of GLuc, NLuc, MLuc7, HTLuc, PaLuc1, PaLuc2, MpLuc1,McLuc1, MaLuc1, MoLuc1, MoLuc2, MLuc39, PsLuc1, LoLuc1-3, HtLuc2,TurboLuc16 (TLuc), Renilla Luc, Firefly luciferase (FfLuc or Fluc),LucPPe-146-1H2, LucPPe-133-1B2, LucPPe-78-0B10, LucPPe49-7C6A,LucPpL-81-6G1 or CBGRluc or homologs or orthologs or mutants orderivatives thereof.

In some embodiments, the target cell has been modified and/or selectedto lack the expression of one or more antigens. In some embodiments, thetarget cell has been modified and/or selected to express one or moreantigens. In an embodiment, the target cell lines is engineered ormodified to express one or more of the SARS-cov2 antigens, epitopes,polyepitopes or fragments thereof. The nucleic acid and amino acid SEQID NO of exemplary SARS-cov2 antigens, epitopes, polyepitopes orfragments are provided in Table 7B. In an embodiment, the target cellline is engineered or modified to express one or more of the SARS-cov2receptors or fragments thereof. Exemplary SARS-cov2 receptor includesACE2 (SEQ ID NO:235 and 705). In an embodiment, the target cell lines isengineered or modified to express one or more genes that enhance theentry and/or replication of SARS-cov2 (e.g. TMPRSS2; SEQ ID NO: 236 and706).

Further provided herein are kits comprising components for assessingcytotoxicity of an agent targeting SARS-cov2. In various embodiments,the kits include target cells (for example, cells engineered to expressintracellularly one or more reporters and/or cell lines expressing oneor more reporters and/or primary cells expressing one or morereporters), and substrates for activating the reporter. The targetcells, reporters and substrates are as described herein. In variousembodiments, the kits further comprise instructions for use of the kit.

Engineered Cell Lines Stably Expressing Thermostable Luciferase forMeasuring Viral Infectivity and Cytopathic Effect.

The disclosure provides cells and cell lines for testing the infectionby viruses (e.g., SARS-cov2) using Matador assays. The method includesexpression of a reporter in target cells in a manner so that it ispreferentially retained within the healthy cells but is either releasedfrom virally infected dead and dying cells or whose activity can bepreferentially measured in dead and dying cells. In one embodiment, theinventive method measures the activity of the reporter that has beenreleased from the dead and dying cells. In some embodiments, thereporters are any one or more of: 1) non-secreted forms of luciferasesfrom the copepods, such as Gaussia princeps, Pleuromamma abdominalis,Metridia pacifica, Metridia curticauda, Metridia asymmetrica, Metridiaokhotensis, Metridia longa, Lucicutia ovaliformis, Heterorhabdustanneri, Pleuromamma scutullata or their homologs; 2) engineeredluciferase reporters from deep sea shrimp, such as NanoLuc; 3) Renillaluciferase; 4) beetle luciferases, including firefly luciferases andengineered variants such as LucPPe-146-1H2. Other reporters, such asGreen Fluorescent Protein, mCherry, and heat-stable alkaline phosphatasemay also be used with the assays described herein. Vectors forexpressing the reporter genes in stable and transient fashion,engineered cell lines stably expressing the reporter genes, and kits forpracticing the invention are also disclosed. In some embodiments, themethods described herein measure the activity of the reporter insidedead and dying cells by addition of a substrate or a cofactor that isrequired for the activity of the reporter and which is excluded from thehealthy cells but preferentially enters dead and dying cells. The methodcan be used to measure the titer of a virus.

Provided herein are methods for assessing infectivity andcytopathogenicity of a virus. The methods include providing a targetcell that is permissive for virus infection and has been engineered toexpress intracellularly a reporter; exposing the target cell to an agentcapable of modulating cytotoxicity; and assaying the activity of thereporter. In one embodiment, a change in reporter activity relative to areference value is indicative of the presence of the virus. In oneembodiment, a change in reporter activity is an increase in reporteractivity relative to a reference value. In some embodiments, thereporter is expressed endogenously by the target cell at a level that isat least 10-25%, 25-50%, 50-75% or 75-100% lower than what is achievedby engineered expression.

In on embodiment, the reporter activity is assayed in the cell mediacontaining the target cells. In another embodiment, the reporteractivity is assayed in the cell supernatant that is free of the targetcells.

In various embodiments, the infectivity of a virus or a preparationcontaining a virus is measured by obtaining (assaying) the reporteractivity in cell pellet and cell free supernatant and normalizing thereporter activity measured in the cell free supernatant. In variousembodiments, normalizing comprises dividing the activity in the cellfree supernatant by the reporter activity measured in the cell pellet.

In various embodiments, the reporter is a non-secretory form of anenzyme that is stable under the assay conditions at 37° C. for more than15 min, for more than 30 min, for more than 1 hour, for more than 2hours, for more than 3 hours, for more than 4 hours, for more than 12hours, 24 hours, for more than 36 hours, for more than 48 hours or forat least 96 hours.

In various embodiments, the reporter is a non-secretory form of aluciferase. In some embodiments, the non-secretory form of luciferase isobtained from copepods, deep sea shrimp, beetle, firefly, or homologs ororthologs thereof or mutants or variants or derivatives thereof. Inexemplary embodiments, the copepods are selected from the groupconsisting of any one or more of Gaussia princeps, Pleuromammaabdominalis, Metridia pacifica, Metridia curticauda, Metridiaasymmetrica, Metridia okhotensis, Metridia longa, Lucicutia ovaliformis,Heterorhabdus tanneri, and Pleuromamma scutullata.

In various embodiments, the luciferase is any one or more of GLuc, NLuc,MLuc7, HTLuc, PaLuc1, PaLuc2, MpLuc1, McLuc1, MaLuc1, MoLuc1, MoLuc2,MLuc39, PsLuc1, LoLuc1-3, HtLuc2, TurboLuc16 (TLuc), Lucia Luc, RenillaLuc, Firefly luciferase (FfLuc or Fluc), LucPPe-146-1H2, LucPPe-133-1B2,LucPPe-78-0B10, LucPPe49-7C6A, LucPpL-81-6G1 or CBGRluc or homologs ororthologs or mutants or variants or derivatives thereof.

In various embodiments, the reporter is a non-secretory form of aluciferase obtained from copepods, deep sea shrimp, beetle, firefly orhomologs or orthologs thereof or mutant or derivatives thereof and thereporter activity is assayed by exposing the target cells to aluciferase specific substrate. In various embodiments, theluciferase-specific substrate is coelentrazine or an imidazopyrazinonesubstrate (furimazine) or a derivative thereof. In one embodiment, theluciferase-specific substrate is D-luciferin or a derivative thereof.

In one embodiment, the reporter is a thermostable luciferase. In oneembodiment, the reporter is a thermostable beetle luciferase. In anembodiment, the thermostable beetle luciferase is obtained from Photurispennsylvanica and Pyrophorus plagiothalamus.

In some embodiments, the reference value is the reporter activity in anyone or more of (i) target cells that do not express reporter, (ii)target cells that express reporter but are not infected with a virus(s); (iii) the target cells that are left untreated; (iv) target cellsthat are not treated with the substrate for the reporter, or (iii) acombination thereof.

In some embodiments, the target cells are exposed to the test agent invitro. In some embodiments, the target cells are exposed to the testagent in vivo. In some embodiments, the assay is performed in vitro. Insome embodiments, the assay is performed in a high throughput fashion.In some embodiments, the assay is performed in vivo.

In an embodiment, the cell line is any cell line that is capable ofsupporting the candidate viral infection. In an exemplary embodiment,the virus is a coronavirus (e.g., SARS-cov2) and the cell line is one ormore of the following, but not limited to, VERO, VERO-E6, HEK-293, BGM,COS, CV-1, FRhK, LLC-MK2, MA-104, MEK, pCMK, HepG2, Huh-7 or RK-13. Insome embodiments, the target cell is a primary cell (e.g., airwayepithelial cells). In an exemplary embodiment, the cells are primaryairway epithelial cells.

In some embodiments, the target cell has been modified and/or selectedto lack or enhance the expression of one or more antigens. The nucleicacid and amino acid SEQ ID NO of exemplary SARS-cov2 antigens, epitopes,polyepitopes or fragments are provided in Table 7B. In an embodiment,the target cell line is engineered or modified to express one or more ofthe SARS-cov2 receptors or fragments thereof. Exemplary SARS-cov2receptor includes ACE2 (SEQ ID NO:235 and 705). In an embodiment, thetarget cell lines is engineered or modified to express one or more genesthat enhance the entry and/or replication of SARS-cov2 (e.g. TMPRSS2;SEQ ID NO: 236 and 706).

Further provided herein are kits comprising components for assessingviral (e.g., SARS-cov2) infectivity and cytopathic effects. In variousembodiments, the kits include target cells (for example, cellsengineered to express intracellularly one or more reporters and/or celllines expressing one or more reporters and/or primary cells expressingone or more reporters), and substrates for activating the reporter.

Topanga Reagent Based on SARS-Cov2 Spike Glycoprotein and NucleocapsidProteins

In various embodiments, the instant invention provides a fast,economical, sensitive and specific Topanga assay and Topanga reagentsfor detection of antibodies, antibody fragments, non-immunoglobulinantigen binding modules (e.g. Centyrins, affibody, DARPINS etc.) andSABR targeting the proteins (e.g. spike glycoprotein and nucleocapsid)encoded by SARS-cov2 that does not require any expensive equipment. Dueto their extreme sensitivity and broad dynamic range, the assay andreagents can be used as a quality-control tool during the manufacturingof SARS-cov2-specific SABR-expressing cells and to monitor theirpersistence in the body after infusion. The Topanga assay and Topangareagents based on the spike glycoprotein and nucleocapsid of SARS-cov2can be also used to detect antibodies, antibody fragments andnon-immunoglobulin antigen binding modules (e.g. Centyrins, affibody,DARPINS etc.) targeting these proteins.

Provided herein is a method for detecting expression of an antibody,antibody fragment, non-immunoglobulin antibody binding module or SABRtargeting Spike glycoprotein of SARS-cov2, comprising: obtaining asample in need determination of expression of the antibody, antibodyfragment, non-immunoglobulin antibody binding module or SABR; optionallyimmobilizing the antibody, antibody, antibody fragment,non-immunoglobulin antibody binding module to a solid surface (e.g.,protein G or anti-IgG or anti-IgM coated plate or beads), contacting thesample with a fusion protein comprising a reporter fused to the spikeglycoprotein of SARS-cov2 or a fragment or variant thereof; washing thesamples to remove any un-bound reporter-Spike glycoprotein fusionprotein, and assaying the activity of the reporter; wherein presence ofreporter activity or increase in reporter activity relative to areference value is indicative of the expression of the antibody,antibody, antibody fragment, non-immunoglobulin antibody binding module,or SABR in the sample. In an embodiment, the Spike glycoprotein isrepresented by SEQ ID NO:701 or a variant thereof having at least 70%homology to the amino acid sequence of SEQ ID NO:701. In an embodiment,the fragment of the Spike glycoprotein comprises its receptor bindingdomain (SEQ ID NO: 700) or a variant thereof having at least 70%homology to the amino acid sequence of SEQ ID NO: 700. The antibody,antibody fragment or the non-immunoglobulin antibody binding module thatbind to the S1-RBD (SEQ ID NO: 700) is likely to have neutralizingactivity against SARS-cov2. In an embodiment, the fragment of spikeglycoprotein comprises its N-terminal domains represented by SEQ ID NO:707 and 712 or variant thereof having at least 70% homology to the aminoacid sequence of SEQ ID NO: 707 and 712. In an embodiment, the fragmentof spike glycoprotein comprises its C-terminal domain (S2 protein)represented by SEQ ID NO: 694 or a variant thereof having at least 70%homology to the amino acid sequence of SEQ ID NO: 694.

Also provided herein is a method for detecting expression of anantibody, antibody fragment, non-immunoglobulin antibody binding moduleor SABR targeting nucleocapsid protein (SEQ ID NO: 702) of SARS-cov2,comprising: obtaining a sample in need determination of expression ofthe antibody, antibody fragment, non-immunoglobulin antibody bindingmodule or SABR; optionally immobilizing the antibody, antibody, antibodyfragment, non-immunoglobulin antibody binding module to a solid surface(e.g., protein G or anti-IgG or anti-IgM coated plate or beads),contacting the sample with a fusion protein comprising a reporter fusedto the nucleocapsid protein of SARSC-cov2 or a fragment or variantthereof; washing the samples to remove any un-boundreporter-nucleocapsid fusion protein, and assaying the activity of thereporter; wherein presence of reporter activity or increase in reporteractivity relative to a reference value is indicative of the expressionof the antibody, antibody, antibody fragment, non-immunoglobulinantibody binding module, or SABR in the sample. In an embodiment, thenucleocapsid protein is represented by SEQ ID NO:702 or variant orfragment thereof having at least 70% homology to the amino acid sequenceof SEQ ID NO:702.

Also provided herein is a method for detecting expression of anantibody, antibody fragment, non-immunoglobulin antibody binding moduleor SABR targeting SARS-cov2 orf3a (SEQ ID NO: 720), comprising:obtaining a sample in need determination of expression of the antibody,antibody fragment, non-immunoglobulin antibody binding module or SABR;optionally immobilizing the antibody, antibody, antibody fragment,non-immunoglobulin antibody binding module to a solid surface (e.g.,protein G or anti-IgG or anti-IgM coated plate or beads), contacting thesample with a fusion protein comprising a reporter fused to the orf3a ofSARSC-cov2 or a fragment or variant thereof; washing the samples toremove any un-bound reporter-nucleocapsid fusion protein, and assayingthe activity of the reporter; wherein presence of reporter activity orincrease in reporter activity relative to a reference value isindicative of the expression of the antibody, antibody, antibodyfragment, non-immunoglobulin antibody binding module, or SABR in thesample. In an embodiment, the orf3a is represented by SEQ ID NO:720 orvariant or fragment thereof having 70% homology to the amino acidsequence of SEQ ID NO:720.

In an embodiment, the sample is a blood sample. In an embodiment, thesample is a serum sample. In an embodiment, the sample is a plasmasample.

In an embodiment, the sample is a fresh sample. In another embodiment,the sample is frozen prior to measurement of the antibody, antibodyfragment or non-immunoglobulin antigen binding module.

In one embodiment, the reporter is a non-secretory form of a luciferase.In exemplary embodiments, the non-secretory form of luciferase isobtained from copepods, deep sea shrimp or homologs or orthologs thereofor mutants or derivatives thereof.

In some embodiments, the copepods are selected from the group consistingof any one or more of Gaussia princeps, Pleuromamma abdominalis,Metridia pacifica, Metridia curticauda, Metridia asymmetrica, Metridiaokhotensis, Metridia longa, Lucicutia ovaliformis, Heterorhabdustanneri, and Pleuromamma scutullata.

In some embodiments, the luciferase is any one or more of GLuc, NanoLuc(NLuc), MLuc7, HtLuc, LoLuc, PaLuc1, PaLuc2, MpLuc1, McLuc1, MaLuc1,MoLuc1, MoLuc2, MLuc39, PsLuc1, LocLuc1-3, HtLuc2 Renilla, TurboLuc16(TLuc) or homologs or orthologs thereof or mutants or functionalderivatives thereof.

In exemplary embodiments, the spike glycoprotein-reporter fusionproteins are represented by SEQ ID NO: 716-718, 1197-1199, 1215-1217,1225-1227, 1232-1234, 1242-1244. These fusion protein constructs alsocarry a C-terminal puromycin resistance module that is separated fromthe spike-glycoprotein-reporter fusion module by a T2A cleavable linker.The puromycin resistance module is optional and is not needed for theactivity of the reporter fusion protein.

In exemplary embodiments, the nucleocapsid-reporter fusion proteins arerepresented by SEQ ID NO: 719, 1211, 1228, 1245 and 1262. These fusionprotein constructs also carry a C-terminal puromycin resistance modulethat is separated from the reporter fusion module by a T2A cleavablelinker. The puromycin resistance module is optional and is not neededfor the activity of the reporter fusion protein.

In exemplary embodiments, the orf3a-reporter fusion proteins arerepresented by SEQ ID NO: 1212, 1229, 1246, and 1263. The constructsalso carry a puromycin resistance module that is optional and is notneeded for the activity of the reporter fusion protein.

In some embodiments, the reporter activity is assayed by addition of aluciferase specific substrate. In one embodiment, theluciferase-specific substrate is coelentrazine or a derivative thereof.In another embodiment, the luciferase-specific substrate isimidazopyrazinone substrate (furimazine) or a derivative thereof.

In an embodiment, the sample is taken from a subject who is at risk ofacquiring SARS-cov2 infection. In an embodiment, the sample is takenfrom a subject who is suspected of having SARS-cov2 infection. In anembodiment, the sample is taken from a subject to determine if thesubject has history of having infected with SARS-cov2. In an embodiment,the sample is taken from a subject to determine if the subject is immuneto SARS-cov2 infection.

In an embodiment, antibody against SARS-cov2 is IgM. In an embodiment,antibody against SARS-cov2 is IgG.

In some embodiments, the SABR is expressed on an immune cell. In oneembodiment, the immune cell is a T cell. In another embodiment, theimmune cell is a CD4 T cell. In a further embodiment, the immune cell isa CD8 T cell. In an embodiment, the immune cell is a Treg cell. In someembodiments, the immune cell is a naive T cell. In some embodiments, theimmune cell is a memory T cell. In some embodiments, the immune cell iscentral memory T cell. In an embodiment, the immune cell is an effectormemory T cell. In an embodiment, immune cell is an NK cell or an NK cellline (e.g., NK92 cell line or NK92MI cell line).

In some embodiments, the SABR is expressed on a stem cell. In anembodiment, the SABR is expressed on a hematopoietic stem cell. In anembodiment, the CAR is expressed on an induced pluripotent stem cell.

In some embodiments, the fusion protein further comprises a tag. Inexemplary embodiments, the tag is any one or more of chitin bindingprotein (CBP), glutathione-S-transferase (GST), polyhistidine (His) tag,FLAG tag, HA tag, Myc tag, V5 tag, AcV5 tag, Streptag or a combinationthereof.

In some embodiments, the reference value is the reporter activity in anyone or more of (i) sample from a subject who has never been exposed toSARS-cov2; (ii) sample from the test subject where the sample is treatedwith an unrelated fusion protein; (iii) samples that are not treatedwith the substrate for the reporter; or (iv) combinations thereof.

In some embodiments, the reference value is the reporter activity in anyone or more of (i) cells that do not express the SABR; (ii) cells thatexpress the SABR but are treated with fusion protein which is not boundby the SABR; (iii) cells that are not treated with the substrate for thereporter; or (iv) combinations thereof.

In some embodiments, the reporter is fused to SARS-cov2 protein (e.g.S1, S2, S1-RBD, nucleocapsid, orf3a) through a covalent bond.

In some embodiments, the reporter is fused to the SARS-cov2 protein(e.g. S1, S2, S1-RBD, nucleocapsid, orf3a) through a non-covalent bond.

In some embodiments, the reporter is fused to the SARS-cov2 protein(e.g. S1, S2, S1-RBD, nucleocapsid, orf3a) through an intermediatemolecule.

In some embodiments, a single molecule of the reporter is fused to theSARS-cov2 protein (e.g. S1, S2, S1-RBD, nucleocapsid, orf3a)

In some embodiments, more than one molecule of the reporter is fused tothe SARS-cov2 protein (e.g. S1, S2, S1-RBD, nucleocapsid, orf3a).

In one embodiment, the assay is performed in vitro. In anotherembodiment, the assay is performed in vivo.

In various embodiments, luciferase activity described herein is measuredby any one or more of methods for measuring light production such as aluminometer, X-ray films, microscopy, or combinations thereof. Thenucleic acid and amino acid sequences of the fusion proteins describedin the above section are presented in the corresponding sequencesections.

The host cell line utilized for expression of the Luc fusion proteins ofthe present invention can be of mammalian or non-mammalian origin. Thoseskilled in the art are credited with ability to preferentially determineparticular host cell lines which are best suited for the desired geneproduct to be expressed therein. In one embodiment, the host cell lineutilized for generation of the Luc fusion proteins of the invention ismammalian in origin. Exemplary host cell lines include, but are notlimited to, DG44 and DUXBll (Chinese Hamster Ovary lines, DHFR minus),HELA (human cervical carcinoma), CVI (monkey kidney line), COS (aderivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line),SP2/0 (mouse myeloma), P3×63-Ag3.653 (mouse myeloma), BFA-lclBPT (bovineendothelial cells), RAJI (human lymphocyte), 293FT and 293 (humankidney). Host cell lines are typically available from commercialservices, the American Tissue Culture Collection or from publishedliterature. In another embodiment, the host cell line utilized forgeneration of the Luc fusion proteins of the invention is of insectorigin. In another embodiment, the host cells utilized for generation ofthe Luc fusion proteins of the invention are bacterial in origin. Inanother embodiment, the host cells utilized for generation of the Lucfusion proteins of the invention are of yeast origin. Those skilled inthe art are credited with ability to preferentially determine particularpromoters and vectors which are best suited for the expression of adesired gene product in a particular host cell line. Exemplary vectorfor expression of the Luc fusion protein of the invention is pLenti-EF1a(SEQ ID NO: 1) or pcDNA3 (SEQ ID NO: 17). Vectors for expression ofproteins in other host cell lines are typically available fromcommercial sources, such as Thermofisher, Novagen and Sigma. In variousembodiments, the fusion proteins described herein may be expressed inbacterial cells, yeasts or insects. The optimum vectors and promoters tobe used for expression of the fusions proteins described herein and forthe methods described herein will be apparent to a person of skill inthe art.

In an embodiment, the fusion proteins are expressed in mammalian cells.In an embodiment, the fusion proteins are expressed in insect cells. Inan embodiment, the fusion proteins are expressed in yeast cells. In anembodiment, the fusion proteins are expressed in bacterial cells. In anembodiment, the fusion proteins are expressed in vitro.

In an embodiment, the assay is conducted with the purified fusionprotein. In an embodiment, the assay is conducted with unpurified fusionprotein.

In an embodiment, the assay is performed as described inPCT/US2017/025602, which is incorporated herein in its entirety.

In an embodiment, the assay is conducted in tubes. In an embodiment, theassay is conducted in microwell plates. In an embodiment, the assay isconducted using Protein G, Protein A or Protein A/G coated plates ormicrobeads. In an embodiment, the assay is conducted using anti-IgG andanti-IgM coated plates or microbeads.

In an embodiment, the assay is carried out in 24 well plates, 48 wellplates, 96 well plates or 384 well plates.

In an embodiment, the assay is used to measure the presence ofantibodies to SARS-cov2 in a sample from a human subject. In anembodiment, the assay is used to measure the presence of antibodies toSARS-cov2 in a sample from an animal subject.

Malibu-Glo Reagents Against SARS-Co2

There are several methods currently in use for detection of SARS-cov2antigens, including Flow cytometry, ELISA and western blotting. However,these methods require expensive equipment (such as flow cytometry),suffer from poor sensitivity and specificity (e.g., ELISA and Westernblotting) or are time consuming. In various embodiments, the instantinvention provides a fast, economical, sensitive and specific assay fordetection of SARS-cov2 infected cells using a non-radioactive antigendetection assay described in PCT/US2017/025602, which is incorporatedherein in its entirety.

Provided herein is a method for detecting the presence of SARS-cov2spike glycoprotein in a sample, comprising: obtaining a sample infectedwith SARS-cov2; optionally immobilizing SARS-cov2 a to a solid surface,contacting the sample with a fusion protein comprising a reporter fusedto moiety (e.g., scFv or vHH fragment etc.) capable of binding the spikeglycoprotein of SARS-cov2; and assaying the activity of the reporter;wherein presence of reporter activity or increase in reporter activityrelative to a reference value is indicative of the expression of thespike glycoprotein in the sample. The nucleic acid and amino acid SEQ IDof exemplary vL, vH and scFv fragments targeting the S1-RBD of SARS-cov2are provided in Table 7A. The SEQ ID NO of the CDRs of the vL, vHfragments comprising the scFv are also provided in Table 7A.

Also provided herein is a method for detecting presence of SARS-cov2 ina sample, comprising: obtaining a sample in need determination ofpresence of SARS-cov2; optionally immobilizing SARS-cov2 to a solidsurface, contacting the sample with a fusion protein comprising areporter fused to the ACE2 extracellular domain (ECD) or a fragment orvariant thereof; washing the samples to remove any un-boundreporter-nucleocapsid fusion protein, and assaying the activity of thereporter; wherein presence of reporter activity or increase in reporteractivity relative to a reference value is indicative of the presence ofSARS-cov2 in the sample. In an embodiment, the ACE2-ECD is representedby SEQ ID NO:705 or variant or fragment thereof.

In one embodiment, the reporter is a non-secretory form of a luciferase.In exemplary embodiments, the non-secretory form of luciferase isobtained from copepods, deep sea shrimp or homologs or orthologs thereofor mutants or derivatives thereof.

In some embodiments, the copepods are selected from the group consistingof any one or more of Gaussia princeps, Pleuromamma abdominalis,Metridia pacifica, Metridia curticauda, Metridia asymmetrica, Metridiaokhotensis, Metridia longa, Lucicutia ovaliformis, Heterorhabdustanneri, and Pleuromamma scutullata.

In some embodiments, the luciferase is any one or more of GLuc, NanoLuc(NLuc), MLuc7, HtLuc, LoLuc, PaLuc1, PaLuc2, MpLuc1, McLuc1, MaLuc1,MoLuc1, MoLuc2, MLuc39, PsLuc1, LocLuc1-3, HtLuc2 Renilla, TurboLuc16(TLuc) or homologs or orthologs thereof or mutants or functionalderivatives thereof.

In an embodiment, the scFv reporter fusion protein targeting the spikeglycoprotein of SARS-cov2 are represented by SEQ ID NO:1202-1204,1218-1221, 1236-1238, 1253-1255 or variants or fragments thereof. Thesefusion protein constructs also carry a C-terminal puromycin resistancemodule that is separated from the reporter fusion module by a T2Acleavable linker. The puromycin resistance module is optional and is notneeded for the activity of the reporter fusion protein.

In an embodiment, the ACE2-ECD reporter fusion protein is represented bySEQ ID NO:1213, 1230, 1247, 1264 or variants or fragments thereof. Thesefusion protein constructs also carry a C-terminal puromycin resistancemodule that is separated from the reporter fusion module by a T2Acleavable linker. The puromycin resistance module is optional and is notneeded for the activity of the reporter fusion protein.

In some embodiments, the reporter activity is assayed by exposing thesample to a luciferase specific substrate. In one embodiment, theluciferase-specific substrate is coelentrazine or a derivative thereof.In another embodiment, the luciferase-specific substrate isimidazopyrazinone substrate (furimazine) or a derivative thereof.

In some embodiments, the fusion protein further comprises a tag. Inexemplary embodiments, the tag is any one or more of chitin bindingprotein (CBP), glutathione-S-transferase (GST), polyhistidine (His) tag,FLAG tag, HA tag, Myc tag, V5 tag, AcV5 tag, Streptag or a combinationthereof.

In some embodiments, the reference value is the reporter activity in anyone or more of (i) sample (e.g., cells or serum) that does not expressthe SARS-cov2 spike glycoprotein; (ii) sample (e.g., cells or serum)that express the SARS-cov2 spike glycoprotein but are treated withfusion protein which does not bind to SARS-cov2 spike glycoprotein;(iii) cells that are not treated with the substrate for the reporter; or(iv) combinations thereof.

In some embodiments, the reporter is fused to the module (e.g., scFv orACE2) targeting SARS-cov2 spike glycoprotein through a covalent bond.

In some embodiments, the reporter is fused to the module (e.g., scFv orACE2) targeting SARS-cov2 spike glycoprotein through a non-covalentbond.

In some embodiments, the reporter is fused to the module (e.g., scFv orACE2) targeting SARS-cov2 spike glycoprotein through an intermediatemolecule.

In some embodiments, a single molecule of the reporter is fused to themodule (e.g., scFv or ACE2) targeting SARS-cov2 spike glycoprotein.

In some embodiments, more than one molecules of the reporter are fusedto the module (e.g., scFv or ACE2) targeting SARS-cov2 spikeglycoprotein.

In an embodiment, the module (e.g., scFv or ACE2) targeting SARS-cov2spike glycoprotein binds to its receptor binding domain.

In one embodiment, the assay is performed in vitro. In anotherembodiment, the assay is performed in vivo.

In an embodiment, the assay is performed as described inPCT/US2017/025602, which is incorporated herein in its entirety.

Also provided herein are kits for practicing the invention.

In another aspect, the invention relates to a kit comprising apharmaceutical composition, cell or cell population, (such as T cells,NK cells, iPSC) comprising vectors encoding nucleic acids encoding SABR,wherein the antigen specific domain of the SABR targets spikeglycoprotein of SARS-cov2.

EXAMPLES

The invention is further described in the non-limiting examples.

Generation of Lentiviral Vectors Encoding SABRs

The sequence of lentiviral vectors pLenti-EF1a or pLenti-EF1α andpLenti-EF1a-DWPRE is represented by SEQ ID NO: 1 and SEQ ID NO: 2. Thesequence of pCCLc-MNDU3 Vector and its variants are provided in SEQ IDNO: 4-6. The generation of Chimeric antigen receptor (e.g., 2^(nd)generation CARs, SIRs, Ab-TCR and TFP etc.) the generation and use ofGGS-NLuc fusion proteins, and the generation and use of luciferase(e.g., GLuc) reporter cell lines for measurement of cellularcytotoxicity using the Matador assays have been described(PCT/US2017/024843, PCT/US2017/025602, PCT/US2017/052344,PCT/US2017/064379 and PCT/US2018/53247), which are incorporated in theirentirety by reference herein.

Example 1: Comparison of SABR Transduction of SARS-Cov2 CTLs Derivedfrom PBMCs and Isolated T Cells

PBMC are collected from a SARS-cov2 sero-positive donor who has beenexposed to SARS-cov2 and recovered from infection as determined byseropositivity for SA cov2 spike glycoprotein or has received aSARS-cov2 vaccine. Optionally, the subject is administered between 1-3booster doses of a SARS-cov2 vaccine between 1-31 days prior tocollection of PBMCs. In another embodiment, PBMC are collected from ahealthy donor who has not been exposed to SARS-cov2. PBMC are thawed andrecovered into RPMI medium. Cells are split into two fractions; ⅓ ofcells (Stimulators) are infected with the lentiviral vector pCCLc-MNDU3(SEQ ID NO: 4) encoding Spike Glycoprotein, membrane glycoprotein,nucleocapsid phosphoprotein and orf3a of SARS-cov2 (nucleic acid SEQ IDNO:1047 and amino acid SEQ ID NO: 1167) by spin-infection. In alternateembodiments, stimulator cells are generated by infection withpCCLc-MNDU3 (SEQ ID NO: 4) encoding different proteins, peptides,polypeptides and polyepitopes of SARS-cov2 either alone, in variouscombinations or in fusion with Ub-G76A mutant. The nucleic acidsequences of exemplary SARS-cov2 proteins, peptides; polypeptides andpolyepitopes are represented by SEQ ID NO:224-234 and 1047-1053 Thecorresponding amino acid sequences are represented by SEQ ID NO: 694-704and 1167-1173, respectively. In an alternate embodiment, the stimulatorcells are generated by infection with an adenoviral vector encodingSpike Glycoprotein, membrane glycoprotein, nucleocapsid phosphoproteinand orf3 a of SARS-cov2. In another alternate embodiment, the stimulatorcells are generated by infection with a lentiviral or adenoviral vectorencoding one or more of the SARS-cov2 proteins, peptides, polypeptidesand polyepitopes in fusion with a drug inducible protein destabilizationdomain (DIPDD), such as FKBP12-F36V (SEQ ID NO: 1161), IKZF1-ZF2-145-167(SEQ ID NO: 1162), IKZF1-ZF2-ZF3-145-197 (SEQ ID NO: 1163),IKZF1-ZF2-ZF3-145-243 (SEQ ID NO: 1164). The nucleic acid sequences ofexemplary SARS-cov2 proteins, peptides, polypeptides and polyepitopes infusion with the DIPDD are represented by SEQ ID NO:1054-1075. Thecorresponding amino acid sequences are represented by SEQ ID NO:1174-1195.

Stimulators are then washed twice and resuspended in RPMI/AB serumculture medium and irradiated at 2500 cGy (2500 rads). The remaining ⅔of cells (Responders) are either transferred to RPMI/AB serum medium andheld at 37° C. until they are be mixed with the Stimulators or are usedto isolate CD3⁺ T cells that are then cultured with the Stimulator PBMC.

In an alternate embodiment, stimulator cells are expanded underconditions that favor the expansion of T cells. For this purpose, afterinfection stimulator cells are resuspended in fresh XVIVO mediacontaining 10 ng/ml CD3 antibody, 10 ng/ml CD28 antibody and 100 IUrecombinant human-IL2. The cells are expanded in GRex flasks for 7-14days to allow the expansion of T cells. Subsequently, the stimulatorcells are irradiated at 2500 cGy (2500 rads) and mixed with responderPBMC or CD3+ T cells.

PBMC Activation

On day 0, cultures are initiated in 6 well (10 cm²) GRex culture platesat ratio of 9×10⁶ irradiated Stimulator PBMC cells to 21×1.0⁷ ResponderPBMC cells and returned to 37° C., tissue culture incubation. Thoughoptional, on days 9 and 10, cell cultures are depleted of CD56⁺, NK(Natural Killer) cells, prior to transduction.

Activation of Isolated CD3⁺T Cells

On day 0, T cells are isolated (i.e., enriched) from PBMC by means knownin the art (e.g., live FACS or anti-CD3-coated magnetic beads). T cellsare initiated in 6 well (10 cm²) GRex culture plates containing mediacomprising 20 U/ml IL2, at a ratio of 1 T cell/4 Stimulator PBMC cellsand returned to 37° C., tissue culture incubation. On days 9 and 10,cell cultures are transduced with the lentiviral vector to express SABRs(as above, NK cell depletion is optional prior to transduction).Sensitizing the starting material (e.g., following CD3⁺ enrichment) toviral antigens, such as SARS-cov2, results in an increase in thepercentage of central memory phenotype T cells in the resultantpopulation.

SABR Transduction

On days 9 and 10 isolated T cell and PBMC cultures are transduced withrecombinant lentivirus encoding anti-CD 19 SABR (and puromycin selectionmarker) using spin-infection as described previously. Briefly,lentivirus encoding anti-CD 19 SABR are thawed at room temperature andadded to plates in the presence of polybrene. Plates are wrapped inparaffin film and centrifuged at 2000×g for 2 hours at 32° C. Viralsupernatant is aspirated and 1 ml of prepared cell suspension from eachstimulation group (0.5×10⁶ cells/mL resuspended in YH5 media) is added.Plates are centrifuged at 1000×g for 15 minutes at 32° C. and incubatedat 37° C., 5% CO2 overnight.

Cells for each stimulation condition are then removed and pooled forcounting. Cells are resuspended in fresh YH5 media at 0.5-1.0×10°cells/ml.

On day 11, each stimulation condition (i.e. of isolated T cells or ofResponder PBMC) are returned to culture with irradiated Stimulator PBMC.Samples are taken at each of Days 15, 23 and 27 forFluorescence-Activated Cell Sorting (FACS) (e.g., for CD3⁺, scFV⁺cells). Drug selection (puromycin) is induced on day 19.

SARS-cov2-CTLs derived from isolated T cells demonstrate improvedviability and proliferative capacity. CD3⁺ enriched starting materialhas a greater yield over conventional expansion and transductionconditions, demonstrating significantly improved viability andproliferative capacity. The efficiency of downstream SABR vectortransduction is dependent upon proliferative capacity. Thus, higherviability and proliferative capacity in the antigen-stimulated T cellsresults in improved downstream SABR transduction efficiency. Whencompared to transduction in a crude PBMC culture, SABR SARS-cov2 CTLsderived from an initial CD3⁺ enrichment step demonstrate a significantenhancement of downstream SABR transduction efficiency post stimulationand also show greater cell viability in response to puromycin selection.

Example 3: Generation of Anti-CD 19-SABR CTL Following Stimulation withPBMC or K562 Cells Expressing One or More of SARS-Cov2 StructuralProteins (e.g., Spike Glycoprotein, Nucleocapsid Phosphoprotein orMembrane Glycoprotein)

Standard anti-CD3/CD28 bead-based stimulation is widely used in thefield as a method to expand T cells in vitro prior to transduction withSABR vectors. The following experiment is undertaken to determine theimpact of various SABR-T cell stimuli amenable to a high-yieldmanufacturing process on the memory T cell immune-phenotype for a finaltherapeutic product.

K562 cells are infected with pCCLc-MNDU3 (SEQ NO: 4) encoding differentproteins, peptides, polypeptides and polyepitopes of SARS-cov2 eitheralone, in various combinations or in fusion with Ub-G76A mutant. Thenucleic acid sequences of SARS-cov2 proteins, peptides, polypeptides andpolyepitopes are represented by SEQ ID NO:224-234 and 1047-1053. Thecorresponding amino acid sequences are represented by SEQ ID NO: 694-704and 1167-1173, respectively. In another alternate embodiment, thestimulator cells are generated by infection with a lentiviral oradenoviral vector encoding one or more of the SARS-cov2 proteins,peptides, polypeptides and polyepitopes in fusion with a drug inducibleprotein destabilization domain (DIPDD), such as FKBP12-F36V (SEQ ID NO:1161), IKZF1-ZF2-145-167 (SEQ ID NO: 1162), IKZF1-ZF2-ZF3-145-197 (SEQID NO: 1163), IKZF1-ZF2-ZF3-145-243 (SEQ ID NO: 1164). The nucleic acidsequences of exemplary SARS-cov2 proteins, peptides, polypeptides andpolyepitopes in fusion with the DIPDD are represented by SEQ IDNO:1054-1075. The corresponding amino acid sequences are represented bySEQ ID NO: 1174-1195. K562 cells are expanded under standard conditions.K562 cells infected with vectors encoding SARS-cov2 proteins, peptides,polypeptides and polyepitopes in fusion with FKBP12-F36V are cultured inthe presence of dTAG13 to induce degradation of the fusion protein. K562cells infected with vectors encoding SARS-cov2 proteins, peptides,polypeptides and polyepitopes in fusion with IKZF1-ZF2-145-167,IKZF1-ZF2-ZF3-145-197, IKZF1-ZF2-ZF3-145-243 are cultured in thepresence of Pomalidomide (2-10 μM), lenalidomide (5-10 μM), CC220 (2-5μM), or CC885 to induce degradation of the fusion proteins.

The K562 cells optionally can be engineered to express one or morecostimulatory molecules, such as 41BBL, CD80, CD83, CD86 and/or vFLIPK13.

The resulting K562 cells are irradiated at 90 Gy (9000 rads) and serveas Stimulator cells as described in the preceding example.

Isolated CD3⁺, SARS-cov2-antigen specific T cells are stimulated andexpanded with no stimulation, stimulation with soluble anti-CD3/CD28,stimulation with anti-CD3/CD28 beads, stimulation with K562 cellsexpressing SEQ ID NO: 1167-1173; stimulation with K562 cells expressingSEQ ID NO: 1186-1190 in the presence of dTAG13; stimulation with K562cells expressing SEQ ID NO: 1191-1195 in the presence of pomalidomide (2μM); stimulation with PBMC expressing SEQ ID NO: 1167-1173; stimulationwith PBMC expressing SEQ ID NO: 1186-1190 in the presence of dTAG13;stimulation with PBMC expressing SEQ ID NO: 1191-1195 in the presence ofpomalidomide (2 μM).

Following 3 days of stimulation, each culture is transduced with a viralvector encoding a synthetic antigen binding receptor (SABR) and returnedto incubation for 7 days in standard (YE media) culture to allow forexpansion of the transduced T cells. Cells are then harvested and cellphenotypes analyzed by cell staining and fluorescence activated cellsorting.

CD3⁺, SARS-cov2-antigen specific T cells are removed from liquidnitrogen storage, thawed, and suspended in YH media at a concentrationof 2×10⁶ cells/mL with 100 IU/mL IL-2. The suspension is plated in a24-well plate with 2 mLs of the suspension per well and incubatedovernight. Following this overnight recovery, cells are counted anddivided into the nine different treatment groups as described above with6×10⁶ cells per group, at a concentration of 1×10⁶ cells/mL.

Stimulation with anti-CD3/CD28 Dynabeads is done at a cell:bead ratio of1:1. A sufficient volume of beads is removed and washed in DPBS prior todirect addition to the cell suspension. Accordingly, stimulation cultureis plated at 2 mL YH5 media with 100 IU/mL IL-2 per well in a 24 wellplate, as above, and incubated for 3 days.

Stimulation with soluble anti-CD3/CD28 is done by direct addition ofCD3/CD28 ImmunoCult™ reagent at a concentration of 25 μl per mL andincubated for 3 days.

Stimulation with SARS-cov2 expressing PBMC is done at a ratio of 1SARS-cov2 antigen-specific T cell to four PBMC. Briefly, CD3+,SARS-cov2-antigen specific T cells are removed from liquid nitrogenstorage, thawed, suspended in YH media, and allowed to recover. Inpreparation for stimulation culture, SARS-cov2-expressing K562 cells areirradiated with a total dose of 90Gy (9000 rads) of radiation.SARS-cov2-K562 and antigen-specific cells are combined in YH5 media with100 IU/mL IL-2 in the same 24-well plate as above and incubated for 3days.

Lentiviral vector encoding the SABR (comprising either a 4-1BB or CD28signal transduction domain) are used to infect the T cells byspin-infection as described.

Cells from each stimulation group are pooled, counted, and resuspendedat a concentration of 0.5-1.0×10⁶ cells/mL of fresh YH5 media beforereturning to incubation at 37° C., 5% CO2 for 2 days. Pooling andresuspension is repeated every two days until the seventh day (harvest).Once harvested cells are labeled and a flow cytometry gating strategy isapplied to identify CD3, CD4, CD8, CD62L and CD45RO on theSABR-expressing I cells. Briefly, cell signals of interest are firstidentified by size and granularity (i.e. forward scatter (FSC) vs. sidescatter (SSC)). Living cells are then identified based on staining witha viability dye (e.g., Live/Dead™ BV510). Viable single cells are thengated based on pulse-area vs. pulse-height (FSC-A vs. FSC-H). IndividualCD3⁺ T cells and subsequent subpopulations (e.g. CD4⁺, CD8⁺, centralmemory T cells) can then be identified using fluorescently-labeledantibodies.

Flow cytometric evaluation indicates that transduced CD 19-SABR-T cellsstimulated with SARS-cov2-K562 or PBMC showed higher percentages ofcentral memory T cell subsets as evaluated by CD62L⁺/CD45RO⁺ cellscompared to soluble CD3/CD28 or bead stimulation.

These data demonstrate that stimulation of SABR-T cell cultures withBLCLs expressing SABR-targeted antigens leads to a shift to predominantcentral memory phenotype when compared to results from CD3/CD28stimulation through bead-bound or soluble antibodies. This studysupports the potential for improved quality of SABR-T cell cultures thatutilize antigen-positive APC stimulation (as represented byantigen-presenting K562 or PBMC) versus the quality expected when usingstandard anti-CD3/CD28 bead or other acellular modes of stimulation.

Example 4: Anti-CD19-SABR-SARS-Cov2-CTLs Exert Potent and SpecificCytotoxicity

Similarly as above, lentiviral vector encoding the SABR (comprisingeither a 4-1BB or CD28 signal transduction domain) is used fortransduction of SARS-cov2-BLCL-stimulated, CD3⁺, antigen-specific Tcells.

Briefly, SABR-expressing, virus-specific T cells are prepared in thefollowing manner.

-   -   Day 0, PBMC samples are thawed, and CD3⁺ cells are enriched        (e.g., by live FACS or anti-CD3-coated magnetic beads). These        CD3⁺ T cells are then stimulated by culturing with        SARS-cov2-antigen-presenting BLCLs, K5462 or T cells as        described herein.    -   11 days post-stimulation (on Day 11), the culture are depleted        of NK cells (e.g., using anti-CD56 beads) and re-stimulated with        fresh SARS-cov2-antigen-presenting BLCLs, K5462 or T cells at a        responder/stimulator ratio of 1:4 for 7 days.    -   On Day 18, the culture are stimulated, at a responder/stimulator        ratio of 4:1 for 2 days.    -   On Day 20 transduction is initiated on with a viral vector        encoding a synthetic antigen binding receptor (SABR) and        returned to incubation in standard (YET media) culture to allow        for expansion of the transduced, virus-specific T cells.        Optionally, an NK cell depletion step may be employed        immediately prior to transduction.    -   By Day 25, SABR-expression is assessed (e.g., by FACS analysis)        and SABR-expressing, virus-specific T cells (effectors) are be        added to target cultures for cytotoxicity assays.

Cytotoxicity, measured by Matador assay, is observed after 4 hours in coculture with SARS-cov2-SABR19-SABR T cells or control SARS-cov2 CTLs atthe ratios of 5:1. SARS-cov2-CD19-SABR T cells demonstrateHLA-independent, CD19-specific cytotoxicity with low allo-cytotoxicityas observed by the specific killing of CD19⁺ (NALM6 and Raji) cells andSARS-cov2⁺/CD19⁺ HLA-matched and mismatched. BLCLs. In contrast, K562cells, and HLA-matched and mismatched PHA blasts, all of which lackSARS-cov2 and CD19 antigen, are not killed. Moreover,SARS-cov2-sensitized, anti-CD 19 SABR T cells are cytotoxic to all CD19⁺cell lines with less off-target cytoxicity, i.e., less or non-existentcytotoxicity to cells lacking both CD19 and SARS-cov2 expression. Whenobserved for three days following effector addition (i.e., SARS-cov2-CTLor SARS-cov2-CD19 SABR T cell addition) specific and potentHLA-independent cytolysis is observed in targeted cells. Cytolysis isinduced in both HLA-matched (BLCL targets) and HLA-mismatched (BLCL andRaji targets) cells. However, SARS-cov2-CTL are shown to only inducesignificant cytolysis in matched BLCL target cells.

When compared to conventionally produced SABR T cells (i.e., withoutenrichment or antigen stimulation of starting T cultures),antigen-specific SABR T cells (e.g., anti-CD19-CD28-SABR-SARS-cov2-CTLs)exhibit comparable, if not better, cytotoxicity. However, anti-CD19-CD28-SABR-SARS-cov2-CTLs appear to be less alloreactive. Theproliferative capacity of SARS-cov2-specific CD 19-SABR T cells isobserved by CellTrace™ Violet dilution assays upon co-culture with theindicated cell lines. Anti-CD19-CD28-SABR-SARS-cov2-CTLs retain theability to kill B-lymphoblastoid cell lines (BLCL) but spared autologousand allogeneic PHA-blast targets lacking CD 19 and SARS-cov2 antigenexpression with notably less alloreactivity relative to conventionalSABR T cells.

Example 5: Use of Allogeneic SABR-Expressing SARS-Cov2 Specific T Cellsfor Adoptive Cells Therapy

Patients with relapsed Acute Lymphocytic Leukemia or high-riskintermediate grade B-cell lymphomas may receive immunotherapy withadoptively transferred HLA-matched allogeneic SABR-T cells. Aleukapheresis product is collected from a SARS-cov2 seropositiveallogeneic donor and used to isolate PBMC by Ficoll-Hypaque separation.A high-resolution HLA typing is done on the donor PBMC. Optionally, CD3positive T lymphocytes are selected using the CliniMACS Prodigy® Systemfrom Miltenyi Biotec and following the manufacturer's recommendations.One third of the T cells are used to generate Stimulator cells, whilethe remainder are used as responder cells. Optionally, the expression ofβ2M and CD52 is eliminated in T cells by CRISPS mediated knock-out usingtechniques known in the art and T cells lacking cell surface expressionof HLA and/or CD52 are selected. PBMC and/or T cells are transduced witha lentiviral vectors encoding SARS-cov2 encoded peptides, polypeptides,proteins and polyepitopes (SEQ ID NO: 1047-1053) to serve as Stimulatorcells. Alternatively, JEKO-1 cells expressing the SARS-cov2 encodedpeptides, polypeptides, proteins and polyepitopes (SEQ ID NO: 1047-1053)and co-expressing 41BBL, CD80, CD83, CD86 and vFLIP K13 serve asStimulator cells. Responder T cells are expanded by co-cultured withStimulator cells at ratio of 1:4 in XVIVO medium with 20 IU/ml of IL2 ina GRex culture flask for 2-7 days to generate SARS-cov2 specific CTLs.The SARS-cov2 specific CTLs are transduced with a clinical gradeCD19-SABR virus (e.g.,CD8SP-hu-mROO5-1-vL-[hTCRb-S57C]-F-P2A-SP-hu-mROO5-1-vH-[hTCRa-T48C](SEQID NO: 261). After viral transduction, SARS-cov2 specific CTLsexpressing CD19 SABR are expanded for 9-12 days in the presence ofirradiated Stimulator JEKO-1 cells or BLCLs in a GRex flask at a ratioof 1:4. After the resulting cell product has undergone quality controltesting (including sterility and tumor specific cytotoxicity tests),they are cryopreserved in aliquotes. A patient with refractory B-All, isenrolled in a trial to receive the allogeneic SARS-cov2 specific CTLsexpressing CD19 SABR. The patient is a full HLA match (12/12) with thedonor. The patient receives lymphodepletive chemotherapy (30 mg/m²/dayfludarabine plus 500 mg/m²/day cyclophosphamide×3 days). The patient mayoptionally receive a single dose of a CD52 antibody (e.g., alemtuzumabat 0.1 to 0.5 mg/kg) between 5-15 days prior to infusion of T cells. Oneday after completion of the lymphodepleting regimen, the patientreceives allogeneic SARS-cov2 specific CTLs expressing CD19 SABRintravenously. The dose of SABR-T product may vary from 1×10⁴ SABR+veCD3 cells/kg to 5×10⁹ SABR+ve CD3 cells/kg as per the study protocol.The SABR product may be administered in a single infusion or splitinfusions. Use of immunosuppressive drugs is at the discretion of thephysician. Essentially a similar approach can be used to treat otherdiseases using allogeneic SARS-cov2 specific CTLs (e.g., T cells)expressing the SABR of the disclosure where the SABR targets an antigenor antigens expressed on the disease causing or disease-associatedcells.

EXAMPLE

SARS-CoV-2-specific T cells were generated from PBMCs isolated from ahealthy donor fully vaccinated with SARS-CoV2 Pfizer vaccine. PBMCs werepulsed with PepTivator SARS-CoV2-Prot_S (Cat #130-126-700; MiltenyiBiotec), a pool of lyophilized peptides, consisting of 15-mer sequenceswith 11 amino acids (aa) overlap, covering the immunodominant sequencedomains of the surface glycoprotein (S) of SARS-CoV2. The PepTivatorSARS-CoV-2 Prot_S contains the sequence domains aa 304-338, 421-475,492-519, 683-707, 741-770, 785-802, and 885-1273. This mix ofoverlapping peptide pool was used at a concentration of 1 μg/μL per15×10⁶ PBMCs for 30 minutes at 37° C. After incubation, cells wereresuspended with 400 IU/mL interleukin-4 (R&D Systems) and 10 ng/mL IL-7(Peprotech) in CTL media consisting of 45% RPMI, 45% Click medium(Irvine Scientific), and 10% fetal bovine serum (FBS) and plated in a6-well GRex plate for expansion. Cytokines and peptide pulsed PBMC werereplenished on day 7. On day 10, cells were harvested and evaluated forantigen specificity and functionality using a SARS-CoV-2 Prot_S T cellanalysis kit (Cat #130-127-586; Miltenyi Biotec).

Expansion is continued as above for 2-3 weeks. After expansion, theSARS-CoV-2 specific T cells are isolated and used to generate SABR-Tcells by infection with a lentiviral vector encoding a SABR againstCD19. The SABR-T cells generated from SARS-CoV2 specific T cells arefound to be less alloreactive in a mixed lymphocytic reaction and causeless Graft vs host disease when tested in immunodeficient mice ascompared to the SABR-T cells generated from parental T cells that havenot been enriched for SARS-CoV2 specificity.

Example 6: Development of Topanga, a Novel Luciferase-Based Assay forDetection of SARS-Cov2 SABR

A challenge in the field of cellular immunotherapy is the lack of arobust assay for detection of SABR on the surface of immune cells. Werecently described a fast, economical, sensitive and specific assay fordetection of SABRs on the surface of immune cells (6). The methodconsists of fusing the antigen binding domain to a marine luciferase.The expression cassette also carries sequences encoding single ormultiple copies of epitope tags, such as FLAG, HA and MYC, Streptag andpolyhistidine tag. To demonstrate the feasibility of the assay, weexpressed a second generation CAR (SEQ ID NO: 309) directed against theS-RBD of SARS-cov2 in JNG cells, which is a clone of Jurkat T cells thathas been engineered to express GFP under an NFAT promoter. S-RBD-NLuc(SEQ ID NO: 717) and PSMA-NLuc (negative control) fusion proteins weregenerated by transient transfection of 293FT cells with thecorresponding expression vectors. The expression cassette for S-RBD-NLuc(SEQ ID NO: 247) also contained a Puromycin resistance gene, which wasseparated from the S-RBD-NLuc cassette by a T2A cleavable linker.Supernatants containing the secreted fusion proteins were collected24-72 h after transfection. JNG-parental (JNG-P) and JNG-S-RBD-BBz CARcells were incubated with S-RBD-NLuc and PSMA-NLuc (negative control)fusion proteins, followed by extensive washes and measurement of thebound fusion protein by luciferase reporter assay. A nearly 20-foldincrease in luciferase activity was observed upon binding of S-RBD-NLucto JNG-S-RBD-BBz CAR cells as compared to the JNG-P cells, demonstratingthe remarkable sensitivity and specificity of the assay. Essentiallysimilar results were obtained when the NLuc was replaced by GLuc, TLucor MLuc7 in the S1-RBD fusion-reporter protein. The nucleic acidsequences of S1-RBD in fusion with the GLuc, TLuc and MLuc7 arerepresented by SEQ ID NO: 1106, 1123 and 1141, respectively. Thecorresponding amino acid sequences are represented by SEQ ID NO:1226,1243 and 1260, respectively.

To demonstrate the feasibility of Topanga assay, we performed a pilotexperiment using SARS-CoV2-monoclonal antibody CR3022 that binds toSARS-CoV2 RBD. Serial dilutions of CR3022 (BEI resources) were added toindividual wells of a Protein G coated 96 well plate in duplicate. Afterincubation, wells were washed and 100 μl of S1-RBD-NLuc supernatant wasadded to each well for 1 h. After washes, luminescence was read byaddition of coelenterazine. An increase in luminescence above the PBScontrol was visible even at the lowest concentration of CR3022 tested(i.e., 0.1 pg/ml), thereby demonstrating the extreme sensitivity of theassay. There was a linear decrease in luminescence with increaseddilution of CR3022 (R2=1).

Serum samples from COVID-19 (+) patients and a normal control wereobtained. Serial dilutions of samples were tested using S1-RBD-NLucsupernatant and using Protein G coated 384 well plate. A differencebetween positive and negative samples was noted even at the lowestdilution (1:1000) tested. To demonstrate the specificity of the assay,14 serum samples from COVID(−) subjects were examined and were found tobe negative.

Topanga-reagent based on SARS-CoV2 RBD was purified using StreptacinResin and demonstrated functional activity against sero-positive andsero-negative samples. Crude supernatant of Topanga Reagents offeredequal sensitivity and specificity as the purified protein. Crude andpurified Topanga reagents also demonstrated increase in immune responseto SARS-CoV2 vaccine. A nearly 2 fold increase in titers were observedafter the first dose of Pfizer vaccine and a nearly 10 fold increase intiter was observed after the 2^(nd) dose of Pfizer vaccine. Essentiallysimilar results are obtained when experiment is repeated using vaccinesfrom other manufacturer (e.g., Moderna, Johnson and Johnson,Astrazaneca, Sinovac etc.)

Next Topanga reagents were generated using S 1-RBD derived fromdifferent variants of SARS-CoV2 containing mutations L452R, L477N,E484K, K417N, K417N-E484K-N501Y etc. The Topanga reagents based onvariants were used to monitor immune response the subject vaccinatedusing the SARS-Cov2 vaccines from Pfizer. It was observed that thevariant Topanga reagents can inform the residual immunity presentagainst the variant in the post-vaccination sample and inform thedecision to receive a booster dose depending on whether the level ofantibodies have fallen below the pre-vaccination level or the level ofantibodies in the control serum.

Example 7: Development of Malibu-Glo Antigen Detection Assay

The antigen binding portion of CARs is generally composed of scFvderived from antibodies. To rapidly screen the different scFv fragmentsand other antigen binding motifs (e.g., vHH, centyrins etc.) for optimalantigen binding affinity, we recently described another novel assaycalled the Malibu-Glo assay. In contrast to the Topanga reagent, theMalibu-Glo reagent involves fusion of an antigen binding motif (e.g., ascFv fragments) to a cassette encoding a marine luciferase (e.g., NLuc)and different epitope tags. To demonstrate the feasibility of theMalibu-Glo assay, we stably expressed the receptor binding domain ofSpike glycoprotein (S-RBD) of SARS-cov2 in K562 cells in a membraneanchored manner by fusion to the hinge and transmembrane domains ofmurine CD8 and generated two mass populations, K562-S-RBD #1 andK562-S-RBD #2, respectively. The nucleic acid and amino acid sequencesof the construct are represented by SEQ ID NO: 229 and 699,respectively. We also generated a lentiviral expression vector encodingthe Malibu-Glo reagentCD8SP-SARScov2-CR3022-(vL-vH)-GGSG-NLuc-4×FLAG-×2STREP-8×His-T2A-PAC(SEQ ID NO:1082). In this vector the scFv targeting the S1-RBD is fusedto a cassette encoding NLuc-4×FLAG-×2STREP-8×His via a GGSG linker. Thecassette is linked via a T2A cleavable linker to a puromycin resistancegene (PAC). We expressed the fusion protein by transient transfection in293FT cells and collected the secreted fusion protein (SEQ ID NO: 1219)in the supernatant approximately 72 h post-transfection. K562-parental(K562-P), K562-S-RBD #1 and K562-S-RBD #2 cells were incubated with thesecreted fusion protein (SARScov2-CR3022-(vL-vH)-GGSG-NLuc). APSMA-GGS-NLuc fusion protein was used as negative control. Followingextensive washes, the cell bound scFv was measured by addition ofcoelentrazine and measurement of light production. A marked increase inluciferase activity was observed upon binding ofSARScov2-CR3022-(vL-vH)-GGSG-NLuc (S-RBD-scFv-NLuc) fusion protein toK562-S-RBD #land K562-S-RBD #2 cells as compared to the K562-P cells. Incontrast, negligible binding of PSMA-GGS-NLuc fusion protein wasobserved to K562-P or K562-S-RBD #1 or #2 cells. These resultsdemonstrate the remarkable sensitivity and specificity of the Malibu-Gloassay for selection of an optimal scFv for incorporation into a CAR. Theresults further demonstrate that the candidate scFv chosen by us canspecifically recognize and bind with good affinity to the receptorbinding domain of the spike glycoprotein (S-RBD) of SARS-cov2.Essentially similar results were obtained when the NLuc module wasreplaced by GLuc, TLuc and MLuc7. The nucleic acid sequences ofSARScov2-CR3022-(vL-vH) in fusion with the GLuc, TLuc and MLuc7 arerepresented by SEQ ID NO: 1099, 1116 and 1133, respectively. Thecorresponding amino acid sequences are represented by SEQ ID NO:1219,1236 and 1253, respectively. Finally, essentially similar results areobtained when the SARScov2-CR3022-(vL-vH) is replaced bySARS-cov2-S-RBD-H4-(vL-vH) and SARScov2-S-RBD-B38-(vL-vH).

Example 8: Generation and Activity of SABR Against SARS-Cov2 SpikeGlycoprotein

Based on the results showing the specific binding and high affinity ofthe scFv SARScov2-CR3022-(vL-vH) against S-RBD, we next incorporatedthis scFv into a lentiviral vector to generate a second-generationS-RBD-specific CAR containing a 41BB costimulatory domain(S-RBD-BBZ-CAR) (SEQ ID NO: 309). We also generated a SABR on the SIRbackground incorporating the vL and vH fragment from this scFv. Thenucleic acid and amino acid sequence of this SIR are represented by SEQID NO: 318 and 788, respectively. We expressed these SABR (CAR and SIR)constructs into JNG cells (Jurkat-NFAT-GFP) cells that express GFP underan NFAT promoter as described above. To test the ability of the SABR toinduce T cell signaling in an antigen-specific manner, we co-culturedthe JNG-P and JNG-S-RBD-BBz CAR and JNG-S-RBD-002-SIR cells withK562-parental (K562-P) and K562-S-RBD and RAJI-parental (RAJI-P) andRAJI-S-RBD cells. Induction of NFAT-signaling driven GFP expression wasmeasured after overnight co-culture using flow-cytometry. Co-culture ofJNG-S-RBD-BBz CAR and JNG-S-RBD-002-SIR cells with K562-S-RBD andRAJI-S-RBD cells led to increase in GFP induction as compared toincubation with K562-P and RAJI-P cells. These results demonstrate thatS-RBD-specific CAR and SIR can specifically recognize and induce T cellsignaling upon encountering target cells expressing cell surfaceexpressed RBD of spike glycoprotein of SARS-cov2. In alternateembodiment, the experiment is repeated with other SABRs targetingSARS-cov2 spike glycoprotein RBD as listed in Table 9-11.

The SABR targeting SARS-cov2 S1-RBD are expressed in primary T cellsusing lentiviral mediated gene transfer. The T cells are expanded andco-cultured with K562-S-RBD and RAJI-S-RBD cells stably expressing GLucfor 24-48 hours. Effective lysis of the target cells is observed usingthe Matador assay. Further, increased secretion of IFNγ and TNFα isobserved in the supernatant of SABR-T cells when cultured withK562-S-RBD and RAJI-S-RBD cells as compared to K562-P and RAJI-P cells.The experiment is also repeated with other SABRs targeting SARS-cov2spike glycoprotein RBD as listed in Table 9-11.

The SABR targeting SARS-cov2 S1-RBD are also expressed in primary Tcells obtained from an allogeneic donor using lentiviral mediated genetransfer. The TRAC and beta2 microglobulin (B2M) genes are deleted inthe donor cells using CRISP/Cas9. The allogeneic T cells are expandedand co-cultured with K562-S-RBD and RAJI-S-RBD cells stably expressingGLuc for 24-48 hours. Effective lysis of the target cells is observedusing the Matador assay. Further, increased secretion of IFNγ and TNFαis observed in the supernatant of SABR-T cells when cultured withK562-S-RBD and RAJI-S-RBD cells as compared to K562-P and RAJI-P cells.The allogeneic SABR-T cells targeting S-RBD of spike glyoprotein ofSARS-cov2 are also administered to a patient with COVID-19. Theexperiment is also repeated with other SABRs targeting SARS-cov2 spikeglycoprotein RBD as listed in Table 9-11.

The SABR targeting SARS-cov2 S1-RBD are also expressed in NK92MI cellline. The cells are expanded under cGMP conditions, tested for potencyusing the Matador assay and administered to a patient with COVID-19 inescalating doses.

The SABR targeting SARS-cov2 S1-RBD are expressed in human iPSC cells.The endogenous TRAC and B2M genes are deleted in the iPSC cells. Thecells are expanded under cGMP conditions and differentiated into NK andT cells in vitro. The cells are tested for potency using the Matadorassay and administered to a patient with COVID-19 in escalating doses.

Example 9: Use of Matador Assay to Test for Presence of SARS-Cov2 andRelated Coronaviruses

VeroE6 cells are engineered to stably express TMPRSS2 (SEQ ID NO: 236).VERO-E6 and VERO-E6-TMPRSS2 cells are further engineered to stablyexpress Glue, NLuc, TLuc, MLuc7 or LucPPe-146-1H2 (SEQ ID NO: 21) inintracelluarly. The SEQ. ID of the luciferases are provided in Table 2.To test the presence of SARS-cov2 in a sample, serial dilution ofsamples are added to VERO-E6 or VERO-E6-TMPRSS2 cells that have beenplated the day before in a 96 well plate to result in 50% confluency.After varying period of time, supernatant is collected and used for themeasurement of luciferase activity by addition of appropriate substrate(e.g., coelentrazine or D-luciferin). In an alternate embodiment, theluciferase activity is directly measured by adding the substrate to the96 well plate as described previously. Cells treated with serialdilution of a known sample of SARS-cov2 serve as positive control,whereas cell treated with phosphate buffer saline serve as negativecontrol. The increase in luciferase activity in sample treated with thevirus as compared to control is reflective of the presence of the virusin the preparation. The titer of the virus is measured by comparison tothe increase in luciferase activity with serially diluted samples ofpositive control.

The above assay is also modified to detect and measure the presence ofan inhibitor of SARS-cov2 infectivity. For example, cells are treatedwith a known dose of SARS-cov2 in the presence of increasingconcentration of a neutralizing antibody to SARS-cov2. The presence ofneutralizing antibody and/or its activity is measured by measuring theluciferase activity in the cell supernatant or in cell population uponthe addition of the appropriate substrate.

In an alternate embodiment, a dual reporter cell line is engineered thatstably expresses intracellularly a marine luciferase (e.g., Gluc, TLuc,NLuc, MLuc7 etc.) and LucPPe-146-1H2 or its variants. After treatmentwith a preparation containing SARS-cov2, the LucPPe-146-1H2 and GLucactivities are sequentially measured from the sample using theDual-Luciferase®Reporter (DLR™) Assay System (Promega) and following themanufacturer's recommendations.

Example 10: Development of Matador Assay to Measure the Cytotoxicity ofCellular and Immunotherapeutics Targeting SARS-Cov2 and RelatedCoronaviruses

K562 and RAJI cells are engineered to stably express Glue, NLuc, MLuc7or LucPPe-146-1H2 (SEQ ID NO: 21) intracelluarly. The cells aresubsequently engineered to express one or more of SARS-cov2 encodedproteins, peptides, polypeptides and polyepitopes as describedpreviously. In an exemplary embodiment, K562-S-RBD and RAJI-S1-RBD cellsare engineered to express Glue intracellularly. To measure thecytotoxicity of a SABR-T cell targeting S-RBD of SARS-cov2, theK562-S-RBD-Glue cells are co-cultured with T cells encoding the SABR(S-RBD-BBZ-CAR) (SEQ ID NO: 309) at an effector to target (E:T) ratio of1:1, 5:1 and 10:1 for 24 hours in a 384 well plate. The cytotoxicity ofSABR-T cells is measured by addition of coelentrazine to the platesusing an autoinjector as described previously.

In alternate embodiment, any suitable cell line expressing a heat stableluciferase and one or more of the SARS-cov2 encoded proteins, peptides,polypeptides and polyepitopes can be used in the Matador assay formeasuring the cytotoxicity of an agent targeting SARS-cov2.

Example 11: Development of Topanga Assay for Detection of SARS-Cov2Antibodies

Expression cassettes encoding Topanga reagents comprising differentprotein encoded by SARS-cov2 in fusion with marine luciferases arerepresented by SEQ ID NO:1197-1200, 1208-1212, 1215-1217, 1225-1229,1232-1234, 1242-1246, 1249-1251 and 1259-1263. The fusion constructs aretransfected in 293FT cells and fusion proteins purified from the cellsupernatant (in case of secreted proteins) and cytosol (in case ofnon-secreted proteins) using StrepTactin column. 96-well Costarflat-bottomed luminometry plates are coated with protein G (5 μg/mL, 100μL/well) in carbonate buffer (pH 9.6) overnight at 4° C. After threewashes with phosphate buffered saline (PBS) containing 0.05% Tween 20(PBS-T), the plates are incubated with blocking solution (PBS containing5% non-fat milk) for 1 h at 37° C. Then the wells are washed, andaliquots of serially diluted sera (100 μL) are added to the wells andincubated for 1 h at 37° C. The plates are washed and incubated with 50μl of diluted Nluc fusion proteins (10-100 ng) for 30 min at 37° C. Thenthe plates are washed again, and luciferase activity is measured afteraddition of coelentrazine. Each sample is tested in duplicate. Thecut-off values are determined as two-fold the average of the normalcontrols. Negative controls are an equal volume mixture of sera fromhealthy blood donors. Results are expressed as the mean RLI (RelativeLuminescence Intensity) from duplicate wells, and values are correctedby subtracting the RLI value of the background wells incubated with HEK293 T cell extracts in the absence of sera. The sensitivity andspecificity of the above assay is found to be superior to a commerciallyavailable ELISA assays (Euroimmun) for measuring SARS-cov2 antibodies.The presence of SARS-cov2 antibodies detected by the receptor bindingdomain of S1 protein is found to correlate with neutralizing antibodiesto SARS-cov2.

In an alternate embodiment, the serum antibodies are captured usingantibodies directed to human IgM or human:IgG subclasses instead of useof Protein G. Thus, for the detection of IgG antibodies againstSARS-cov2, the plate is coated overnight with goal anti-Human IgGantibody. Similarly, for the detection of IgM antibodies againstSARS-cov2, the plate is coated overnight with goal anti-Human IgMantibody. After three washes with phosphate buffered saline (PBS)containing 0.05% Tween 20 (PBS-T), the plates are incubated withblocking solution (PBS containing 5% non-fat milk) for 1 h at 37° C.Then the wells are washed, and aliquots of serially diluted sera (100μL) are added to the wells and incubated for 1 h at 37° C. The platesare washed and incubated with 50 μl of diluted marine luciferase fusionproteins (10-100 ng) for 30 min at 37° C. Then the plates are washedagain, and luciferase activity is measured after addition ofcoelentrazine. The sensitivity and specificity of the above assay isfound to be superior to a commercially available ELISA assays formeasuring SARS-cov2 antibodies. The presence of SARS-cov2 antibodiesdetected by the receptor binding domain of S1 protein is found tocorrelate with neutralizing antibodies to SARS-cov2.

All publications, patents, patent applications and sequence accessionnumbers mentioned herein are hereby incorporated by reference in theirentirety as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims,

What is claimed is:
 1. A method of generating a composition comprisingcytotoxic T cells that are specific for binding to a SARS-cov2 encodedfirst antigen and that express a synthetic antigen binding receptor(SABR) specific for binding to a second antigen, said method comprising:i) preparing a culture of cells enriched for CD3⁺ cells, said CD3⁺ Tcells being stimulated to recognize and bind to said SARS-cov2 encodedfirst antigen through a T-cell receptor; ii) transducing the CD3⁺ Tcells of said culture with a viral vector comprising a nucleic acidsequence encoding a SABR that binds to said second antigen; iii)culturing the SABR-transduced CD3⁺ T cells to allow proliferation ofSABR-expressing, first-antigen-specific cytotoxic T cells; and iv)harvesting the SABR-expressing, first-antigen-specific cytotoxic Tcells.
 2. A method of preparing an adoptive immunotherapy compositioncomprising cytotoxic T cells that are specific for binding to aSARS-cov2-encoded first antigen and that express a synthetic antigenbinding receptor (SABR) specific for binding to a second antigen, saidmethod comprising: i) preparing a culture of cells enriched for CD3⁺ Tcells, said CD3⁺ T cells being stimulated to recognize and bind to saidSARS-cov2-encoded first antigen through a T-cell receptor; ii)transducing the CD3⁺ T cells of said culture with a viral vectorcomprising a nucleic acid sequence encoding a SABR that binds to saidsecond antigen; iii) culturing the SABR-transduced CD3⁺ T cells to allowproliferation of SABR-expressing, SARS-cov2 encodedfirst-antigen-specific cytotoxic T cells; and iv) harvesting theSABR-expressing, SARS-cov2-encoded first antigen-specific cytotoxiccells, thereby providing said adoptive immunotherapy composition.
 3. Amethod for inducing proliferation of a population of cytotoxic T cellsthat are specific for binding to a SARS-cov2-encoded first antigen andthat express a chimeric antigen receptor (SABR) specific for binding toa second antigen, said method comprising: i) preparing a culture ofcells enriched for CD3⁺ T cells, said CD3⁺ T cells being stimulated torecognize and bind to said SARS-cov2-encoded first antigen through aI-cell receptor; ii) transducing the CD3⁺ T cells of said culture with aviral vector comprising a nucleic acid sequence encoding a SABR thatbinds to said second antigen; and iii) culturing the SABR-transducedCD3⁺ T cells to allow proliferation of SABR-expressing,first-antigen-specific cytotoxic T cells.
 4. The method of claim 1,further comprising incubating the culture of steps i), ii), iii), or anycombination thereof, with one or more cytokines.
 5. The method of claim1, wherein the culture of cells enriched for CD3⁺ cells comprises asample of peripheral blood mononuclear cells (PBMCs) depleted of redblood cells, platelets, monocytes, and granulocytes.
 6. The method ofclaim 1, wherein the culture of cells enriched for CD3⁺ cells isprepared by a process comprising positive selection of CD3⁺ cells from asample of PBMCs
 7. The method of claim 1, wherein the step of preparinga culture of cells enriched for CD3⁺ T cells stimulated to recognize andbind to said SARS-cov2-encoded first antigen through a T-cell receptor,comprises co-culturing a responder population of CD3⁺ cells withstimulator cells that present said SARS-cov2-encoded first antigen ontheir cell surface.
 8. The method of claim 7, wherein said co-culturingis initiated at a responder cells:stimulator cells ratio of 1:4.
 9. Themethod of claim 7, wherein said prepared culture of cells enriched forCD3⁺ T cells stimulated to recognize and bind to said SARS-cov2-encodedfirst antigen through a I-cell receptor is optionally re-cultured withstimulator cells that present said SARS-cov2-encoded first antigen ontheir surface prior to SABR transduction.
 10. The method of claim 9,wherein a first re-culture step is initiated at least 7 days afterinitiation of the co-culture step.
 11. The method of claim 7, whereinthe responder and stimulator cells are derived from the same donor. 12.The method of claim 7, wherein the stimulator cells are gamma irradiatedantigen-presenting stimulator cells.
 13. The method of claim 7, whereinthe stimulator cells comprise lymphoblastoid B cells (BLCLs), T cells orantigen presenting cell line (e.g., K562, REC-1, JEKO-1, GENTA etc.).14. The method of claim 7, wherein the stimulator cells are infectedwith a native and/or wild-type SARS-Cov2 virus or variant comprising atleast one immunogenic peptide antigen comprising a T cell epitope). 15.The method of claim 7, wherein the stimulator cells have been engineeredto express at least one immunogenic peptide antigen comprising a T cellepitope.
 16. The method of claim 1, wherein the SARS-cov2-encoded firstantigen is a viral antigen.
 17. The method of claim 16, wherein theviral antigen is SARS-cov2 Spike Glycoprotein peptide or fragmentthereof, an SARS-cov2 nucleocapsid phosphoprotein peptide or fragmentthereof, SARS-cov2 membrane glycoprotein peptide or fragment thereof,SARS-cov2 encoded orf3a or fragment thereof, SARS-cov2 encoded orflab orfragment thereof or a variant of the above.
 18. The method of claim 16,wherein the viral antigen comprises a peptide, polypeptide orpolyepitope derived from SARS-cov2 Spike Glycoprotein peptide orfragment thereof, an SARS-cov2 nucleocapsid phosphoprotein peptide orfragment thereof, SARS-cov2 membrane glycoprotein peptide or fragmentthereof, SARS-cov2 encoded orf3a or fragment thereof, SARS-cov2 encodedorflab or fragment thereof.
 19. The method of claim 7, wherein thestimulator cells further express said second antigen.
 20. The method ofclaim 1, wherein said second antigen is any one or more of but notlimited to CD5; CD1.9; CD123; CD22; CD30; CD171; CS1 (also referred toas CD2 subset 1, CRACC, MPL, SLAMF7, CD319, and 19A24); C-typelectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factorreceptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms Like Tyrosine Kinase3 (n173); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; aglycosylated CD43 epitope expressed on acute leukemia or lymphoma butnot on hematopoietic progenitors, a glycosylated CD43 epitope expressedon non-hematopoietic cancers, Carcinoembryonic antigen (CEA); Epithelialcell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117);Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2);Mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cellantigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascularendothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24;Platelet-derived growth factor receptor beta (PDGFR-beta);Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha(FRa or FR1); Folate receptor beta (FRb); Receptor tyrosine-proteinkinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1);epidermal growth factor receptor (EGFR); neural cell adhesion molecule(NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (PAP);insulin-like growth factor 1 receptor (IGF-I receptor), carbonicanhydrase IX (CAlX); Proteasome (Prosome, Macropain) Subunit, Beta Type,9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consistingof breakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3(aNeu5Ac(2-3)bDClalp(l-4)bDGlcp(l-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); tumor endothelial marker 1 (TEM1/CD248); tumorendothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroidstimulating hormone receptor (TSHR); G protein coupled receptor class Cgroup 5, member D (GPRC5D); chromosome X open reading frame 61(CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialicacid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoHglycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1);uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1);adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupledreceptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K);Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading FrameProtein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1(NY-ESO-1); Cancer/testis antigen 2 (LAGS-1a); Melanoma-associatedantigen 1 (MAGE-A1); ETS translocation-variant gene 6, located onchromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family,Member 1A (XAGEl); angiopoietin-binding cell surface receptor 2 (Tie 2);melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testisantigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53);p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumorantigen-1 (PCT A-1 or Galectin 8), melanoma antigen recognized by Tcells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerasereverse transcriptase (hTERT); sarcoma translocation breakpoints;melanoma inhibitor of apoptosis (MIL-IAP); ERG (transmembrane protease,serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V(NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl;v-myc avian myelocytomatosis viral oncogene neuroblastoma derivedhomolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-relatedprotein 2 (TRP-2); Cytochrome P450 lB 1 (CYPlB 1); CCCTC-Binding Factor(Zinc Finger Protein)-Like (BORIS or Brother of the Regulator ofImprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding proteinsp32 (OY-TESl); lymphocyte-specific protein tyrosine kinase (LCK); Akinase anchor protein 4 (AKAP-4); synovial sarcoma; X breakpoint 2(SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renalubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinalcarboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a;CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1(LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyteimmunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300molecule-like family member f (CD300LF); C-type lectin domain family 12member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-likemodule-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyteantigen 75 (LY75); Glypican-3 (GPC3); Fe receptor-like 5 (FCRL5); andimmunoglobulin lambda-like polypeptide 1 (IGLL1), MPL, Biotin, c-MYCepitope Tag, CD34, LAMP1 TROP2, GFRalpha4, CDH17, CDH6; NYBR1, CDH19,CD200R, Slea (CA19.9; Sialyl Lewis Antigen); Fucosyl-GM1, PTK7, gpNMB,CDH1-CD324, DLL3, CD276/B7H3, IL13Ra2, CD179b-IGLl1, TCRgamma-delta,NKG2D, CD32 (FCGR2A), Tn ag, Tim1-/HVCR1, CSF2RA (GM-CSFR-alpha),TGFbetaR2, Lews Ag, TCR-beta1 chain, TCR-beta2 chain, TCR-gamma chain,TCR-delta chain, FITC, Leutenizing hormone receptor (LHR), Folliclestimulating hormone receptor (FSHR), Gonadotropin Hormone receptor (CGHRor GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein,HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza Ahemagglutinin (HA), GAD, PDL1, Guanylyl cyclase C (GCC), auto antibodyto desmoglein 3 (Dsg3), auto antibody to desmoglein 1 (Dsg1), HLA,HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ,HLA-DR, HLA-G, IgE, CD99, Ras G12V, Tissue Factor 1 (TF1), AFP, GPRC5D,Claudin18.2 (CLD18A2 or CLDN18A.2), P-glycoprotein, STEAP1, Liv1,Nectin-4, Cripto, gpA33, BST1/CD157, low conductance chloride channel,the antigen recognized by TNT antibody; CD229, Toso, BAFF-R, receptorbinding domain of spike glycoprotein of SARS-cov2.